Pulsed-multiline excitation for color-blind fluorescence detection

ABSTRACT

The present invention provides a technology called Pulse-Multiline Excitation or PME. This technology provides a novel approach to fluorescence detection with application for high-throughput identification of informative SNPs, which could lead to more accurate diagnosis of inherited disease, better prognosis of risk susceptibilities, or identification of sporadic mutations. The PME technology has two main advantages that significantly increase fluorescence sensitivity: (1) optimal excitation of all fluorophores in the genomic assay and (2) “color-blind” detection, which collects considerably more light than standard wavelength resolved detection. This technology differs significantly from the current state-of-the-art DNA sequencing instrumentation, which features single source excitation and color dispersion for DNA sequence identification. Successful implementation of the PME technology will have broad application for routine usage in clinical diagnostics, forensics, and general sequencing methodologies and will have the capability, flexibility, and portability of targeted sequence variation assays for a large majority of the population.

BACKGROUND OF THE INVENTION

[0001] I. Field of the Invention

[0002] The present invention relates generally to the fields of highthroughput genetic analysis applications and fluorescence spectroscopy.More particularly, it provides a variety of compositions and methods foruse in high-throughput DNA sequence identification.

[0003] II. Description of Related Art

[0004] The Human Genome Project (HGP) holds tremendous promise fordiscoveries of the molecular mechanisms that trigger the onset of manycommon diseases over the next several decades. The initial HGP goalsunderway will provide or have provided the complete and accurate genomesequences of human and multiple well-studied genetic model organisms,such as mouse, rat, fruit fly, nematode, yeast and numerous bacteria.From this foundation of reference genome sequences, the elucidation ofcomplete gene sets, coupled with comparative cross-species studies, areexpected to assist significantly in the assignment to specific humangenes of protein function and disease associations. Other technologiescomplement the assignment of biological functions: gene and proteinexpression profiling, mouse gene-knockouts, and techniques that measureprotein-protein interactions. The elucidation of gene structure-proteinfunction relationships are key to understanding how genomic sequencevariation between individuals can cause increased risk or predispositionto certain complex diseases or are even the etiologic agents responsiblefor the onset of particular diseases. However, the use of geneticvariation in clinical practice is only beginning and technology tofacilitate its use is greatly needed.

[0005] The most commonly observed form of human sequence variation issingle nucleotide polymorphisms (SNPs), which occur at a frequency ofapproximately 1-in-300 to 1-in-1000 base pairs. In general, 10%-to-15%of SNPs will affect either protein function by altering specific aminoacid residues, or will affect the proper processing of genes by changingsplicing mechanisms, or will affect the normal level of expression ofthe gene or protein by varying regulatory mechanisms. Several recentexamples are the associations of mutations with the NOTCH4 gene andschizophrenia (Wei et al., 2000), peroxisome proliferator-activatedreceptor gamma (PPARγ) gene and severe insulin resistance (Deeb et al.,1998), and melanocortin-4 receptor (MC4R) gene and inherited obesity(Yeo et al., 1998).

[0006] The identification of informative SNPs will lead to more accuratediagnosis of inherited diseases, better assessment of risksusceptibilities, and could be assayed in specific tissue biopsies forsporadic mutations. An individual's SNP profile could be used to offsetand significantly delay the progression of disease by helping in thechoice of prophylactic drug therapies. A SNP profile of drugmetabolizing genes could be used to prescribe a specific drug regimen toprovide safer and more efficacious results. To accomplish goals likethese, genome sequencing will move into the resequencing phase of notjust a handful of individuals, but potentially the partial sequencing ofmost of the population. Resequencing simply means sequencing in parallelspecific regions or single nucleotides that are distributed throughoutthe human genome to obtain the SNP profile for a given complex disease.

[0007] For this technology to be applicable and practicable for routineusage in medical practice, it must be robust, easy-to-use, highlysensitive, flexible, portable, and the results should be accurate andrapidly obtained. While current technologies at large genome centers arerobust and results are accurate, they are inadequate and inflexible forresequencing millions of individuals in routine clinical practice. It istherefore advantageous to develop a DNA sequencing instrument, whichmeets these needs. Miniaturization of this technology is alsoadvantageous because smaller instruments potentially require less sampleand reagents and can be more readily transported and located in areassuch as clinics or doctors' offices.

[0008] Ideally, DNA sequencing technology would have the sensitivity fordirect assays without DNA amplification, and be simple and portable forroutine usage in basic, applied, and clinical laboratories. Currently,DNA sequencing technology for high-throughput analyses are specializedand centralized in large genome centers and require numerous molecularbiology manipulations that take days or weeks of preparation before DNAsequence analysis can be performed. Thereafter, the state-of-the-arttechnology involves the attachment of four different fluorescent dyes orfluorophores to the four bases of DNA (i.e., A, C, G, and T) that can bediscriminated by their respective emission wavelengths, theelectrophoretic separation of the nested set of dye-labeled DNAfragments into base-pair increments, and the detection of the dyefluorescence following irradiation by a single argon-ion laser source.Current instrumentation for electrophoretic separation comprises a96-capillary array that disperses the different fluorescent signalsusing a prism, diffraction grating, spectrograph, or other dispersingelement and images the four colors onto a charged-coupled device (CCD)camera. The throughput of each 96-capillary instrument is approximately800 DNA samples per day, and the success of the HGP in large-scalegenomic sequencing has been attributed to the use of hundreds of thesemachines throughout the world. The main disadvantages of the currenttechnology are the laborious cloning or amplification steps needed toprovide sufficient DNA material for analyses, the relatively large sizeof the instruments (roughly the size of a 4-foot refrigerator), and theinadequate sensitivity of detection (i.e., inefficient excitation offluorescent dyes with absorption maxima far from the laser excitationwavelength).

[0009] Although the resolution of spectral emission wavelengths is themainstream technology used in commercial and academic prototypeinstruments, several groups have explored other physical properties offluorescence as a method for discriminating multicolor systems for DNAsequence determination. Recently, Lieberwirth et al. (1998) described adiode-laser based time-resolved fluorescence confocal detection systemfor DNA sequencing by capillary electrophoresis. In this system, asemiconductor laser (630 nm) was modulated using a tunable pulsegenerator at a repetition rate of 22 MHz (454 psec pulses) and focusedby a microscope objective. The fluorescence was collected by the sameobjective and imaged on a single photon counting module APD (Lieberwirthet al., 1998).

[0010] The Luryi group at SUNY Stony Brook have proposed a multiplelaser excitation approach using different radio frequency (RF)modulations and demodulations to discriminate a mixture of fluorophores(U.S. Pat. Nos. 5,784,157 and 6,038,023). U.S. Pat. No. 5,784,157describes a 4-laser based fiber optic single capillary monitoringdevice, which initially has a non-wavelength component, but later theinvention discusses the coupling of spectral resolution for fluorophorediscrimination. There are three significant flaws apparent in thissystem relating to the enhanced fluorescence cross-talk and laserscattered light, low sensitivity detection, and a system that does notappear to scale beyond one capillary.

[0011] As described, the target capillary is illuminated simultaneouslyby all four lasers, which are modulated by different RF signals. Thedifferent RF signals for all of the dyes are summed together and thedetector photodiodes are demodulated by additional heterodyne RFsignals. Interestingly, Gorfinkel and Luryi describe the creation ofBragg reflectors to eliminate cross-talk modulation for a given dye set.Fluorescence cross-talk, however, will not be eliminated using thistechnique. Signal from the “wrong” dye, which is weakly excitedoff-resonance by a particular laser, will be encoded with thecorresponding “wrong” frequency, decoded, and added to the signal forthe target dye. Moreover, scattered laser light will also be modulated,and is likewise not rejected by the heterodyne detection.

[0012] The simultaneous multi-modulation method also has a seriousshortcoming for the detection of low light levels, which is a specificaim of the current invention. All the lasers are proposed to operatesimultaneously, followed by detection of substantially all of the entirefluorescence, and conversion of the collected fluorescence to anelectrical signal. This design potentially creates a correspondinglyhigh quantum statistical noise level, which should be distributed to allthe detectors. The demultiplexing process of RFs does not remove thisexcessive random noise, even if the corresponding signal is small(Meaburn, 1976). In comparison, the Pulse-Multiline Excitation (PME)system described in the current invention exhibits noise levels inproper proportion, so that a weak signal originating from a particularlaser pulse has a correspondingly low detected noise level during thatlaser's sub-cycle. Optimizing the optical system for producing low noiselevels is essential in establishing the optimum contrast between thepresence and absence of a given dye.

[0013] Finally, U.S. Pat. No. 5,784,157 describes a rather complicatedarray of optical fibers, combiners, splitters, and 4 heterodynedetectors with their associated spectral filters for a single capillarychannel. Scaling this system to a 2-capillary system would entaildoubling the mentioned detector components. Unfortunately a CCD camerais not readily adapted for high frequency RF modulation, as it is an“inherently discrete-time” device. In a more recent document, U.S. Pat.No. 6,038,023, the multiplicity of spectral filters has been replacedwith a dispersing prism spectrometer and a high speed one dimensionalarray detector for use with a single capillary channel device; thepotential to scale up to a capillary array system is more feasible asdiscussed by the Luryi group, but may require a multiplicity of suchspectrometer units.

[0014] The current invention comprises a novel fluorescence device,which is capable of significant improvements in the limit of detectionof multi-color fluorescence reactions and may be applied to directmeasurement of such reactions from biological sources (i.e., without theneed for PCR or cloning amplifications). Moreover, this technology,called Pulse-Multiline Excitation or “PME” can be configured on a smallwork surface or in a small instrument, compared to the current DNAsequencing instruments. Thus, a DNA sequencer the size of a suitcase orsmaller is described.

[0015] The development of improved DNA sequencing chemistries willlikely improve the number of independent assays that can be run inparallel. This technology will have broad application in both generalsequencing and forensic applications.

SUMMARY OF THE INVENTION

[0016] Thus, the present invention contemplates an apparatus and methodfor use in high-throughput DNA sequence identification. An aspect of theinvention is a pulse-multiline excitation apparatus for analyzing asample containing one or more fluorescent species, comprising: one ormore lasers configured to emit two or more excitation lines, eachexcitation line having a different wavelength; a timing circuit coupledto the one or more lasers and configured to generate the two or moreexcitation lines sequentially according to a timing program to producetime-correlated fluorescence emission signals from the sample; anon-dispersive detector positioned to collect the time-correlatedfluorescence emission signals emanating from the sample; and an analyzercoupled to the detector and configured to associate the time-correlatedfluorescence emission signals with the timing program to identifyconstituents of the sample.

[0017] The detector and the analyzer may be integral. In one embodiment,the two or more excitation lines intersect at the sample, or the two ormore excitation lines may be configured so that they do not intersect inthe sample. The two or more excitation lines may be coaxial.

[0018] In one embodiment of the invention, the apparatus may furthercomprise an assembly of one or more prisms in operative relation withthe one or more lasers and configured to render radiation of the two ormore excitation lines substantially colinear and/or coaxial.

[0019] The apparatus may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16 or more excitation lines having 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or more excitation wavelengths, respectively.The sample may be comprised in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, up to 20, up to 24, up to 28, up to 36, up to 48, up to64, up to 96, up to 384 or more capillaries. A sheath flow cuvette maybe used.

[0020] The timing program may comprise a delay between the firing ofeach laser of between about 10 fs and about 5 s, between about 1 ms andabout 100 ms, or between about 50 ps and about 500 ps. One or more ofthe excitation lines is pulsed. The pulsed excitation line may becontrolled by TTL logic or by mechanical or electronic means. In oneembodiment, the apparatus may generate a sequence of discrete excitationlines that are time-correlated with the fluorescence emission signalsfrom the sample.

[0021] The lasers may independently comprise a diode laser, asemiconductor laser, a gas laser, such as an argon ion, krypton, orhelium-neon laser, a diode laser, a solid-state laser such as aNeodymium laser which will include an ion-gain medium, such as YAG andyttrium vanadate (YVO₄), or a diode pumped solid state laser. Otherdevices, which produce light at one or more discrete excitationwavelengths, may also be used in place of the laser. The laser mayfurther comprise a Raman shifter in operable relation with at least onelaser beam. In one embodiment of the invention, the excitationwavelength provided by each laser is optically matched to the absorptionwavelength of each fluorophore.

[0022] The detector may comprise a charged couple device, aphotomultiplier tube, a silicon avalanche photodiode or a silicon PINdetector. The footprint of the device is preferably small, such as lessthan 4 ft×4 ft×2ft, less than 1 ft×1ft×2ft, and could be made as smallas 1 in×3 in×6 in.

[0023] Another aspect of the current invention comprises a method ofidentifying sample components comprising: (a) preparing a samplecomprising sample components, a first dye and a second dye; (b) placingthe sample in the beam path of a first excitation line and a secondexcitation line; (c) sequentially firing the first excitation line andthe second excitation line; (d) collecting fluorescence signals from thesamples as a function of time; and (e) sorting the fluorescence by eachexcitation line's on-time window, wherein the sample components areidentified. It is an aspect of the invention that the fluorescencesignals are collected from discrete time periods in which no excitationline is incident on the sample, the time periods occurring between thefiring of the two excitation lines. This technique is known as “lookingin the dark.” Yet another aspect of the present invention is that theabsorption maximum of the first dye substantially corresponds to theexcitation wavelength of the first excitation line. The absorptionmaximum of the second dye may also substantially corresponds to theexcitation wavelength of the second excitation line. In yet anotheraspect of the current invention there is a third and fourth dye and athird and fourth excitation line, wherein the absorption maxima of thethird and fourth dyes substantially correspond to the excitationwavelengths of the third and four excitation lines, respectively.Similarly, there may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ormore dyes wherein the absorption maxima of the dyes substantiallycorresponds to excitation wavelengths of a 5^(th), 6^(th), 7^(th),8^(th), 9^(th), 10^(th), 11^(th), 12^(th), 13^(th), 14^(th), or moreexcitation lines, respectively. The dyes may be a zanthene, fluorescein,rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine,phycobiliprotein, Alexa, squariane dyes, or some other suitable dye.

[0024] In one embodiment of the current invention, the sample componentsenable the determination of SNPs. The method may be for thehigh-throughput identification of informative SNPs. The SNPs may beobtained directly from genomic DNA material, from PCR amplifiedmaterial, or from cloned DNA material and may be assayed using a singlenucleotide primer extension method. The single nucleotide primerextension method may comprise using single unlabeled dNTPs, singlelabeled dNTPs, single 3′-modified dNTPs, single base-modified 3′-dNTPs,single alpha-thio-dNTPs or single labeled 2′,3′-dideoxynucleotides. Themini-sequencing method may comprise using single unlabeled dNTPs, singlelabeled dNTPs, single 3′-modified dNTPs, single base-modified 3′-dNTPs,single alpha-thio-dNTPs or single labeled 2′,3′-dideoxynucleotides. TheSNPs may be obtained directly from genomic DNA material, from PCRamplified material, or from cloned DNA materials and may be assayedusing Sanger sequencing.

[0025] In another embodiment of the current invention, analyzing thesignals is adapted for the accurate diagnosis of inherited disease,better prognosis of risk susceptibilities, identification of sporadicmutations, or prescribing tailor-made daily drug regimens for individualpatients. Analyzing the signals may be adapted for routine usage inclinical diagnostics, forensics applications or determining generalsequencing methodologies.

[0026] Yet another aspect of the current invention is a method ofidentifying sample components comprising: (a) obtaining a biologicalsample; (b) labeling said sample with one or more fluorophores; (c)separating components of said sample; and (d) detecting said samplecomponents with a device wherein said device may comprise: one or morelasers configured to emit two or more excitation lines, each excitationline having a different excitation wavelength; a timing circuit coupledto the one or more lasers and configured to fire the two or moreexcitation lines sequentially according to a timing program to producetime-correlated fluorescence emission signals from the sample; and anon-dispersive detector positioned to collect the time-correlatedfluorescence emission signals; wherein said detector collects timecorrelated data from said sample comprising fluorescent emissions of thesample as a result of irradiation by the one or more excitation lines.

[0027] The sample components may be nucleic acids, amino acids orproteins. The separation may be by electrophoresis, chromatography ormass spectrometry (MS) such as MALDI-TOF, quadrapole mass filter ormagnetic sector MS. The sample components may be addressed on highdensity chip arrays.

[0028] In one embodiment, the method may further comprise: (e)contacting said sample components on a surface comprising immobilizedoligonucleotides at known locations on said surface; and (f) performinga single nucleotide incorporation assay or a mini-sequencing assay. Inyet another embodiment, the method may further comprise rastering saidsurface or said excitation lines such that said excitation lines contactsaid surface at multiple locations.

[0029] Another aspect of the current invention is a device comprising:(a) one or more lasers having two or more excitation lines; (b) one ormore beam steering mirrors wherein said excitation lines each strikesaid mirrors; (c) a first prism, wherein said two or more excitationlines strike one surface and exit from a second surface of said firstprism; and (d) a second prism at an angle relative to said first prism,wherein said two or more excitation lines strike one surface of saidsecond prism after exiting said first prism and exit said second prism,wherein said two or more excitation lines are substantially colinearand/or substantially coaxial after exiting said second prism. The angleof the second prism relative to the first prism is dependent on theoptical material used. For example, for high dispersion flint glass, thetwo prisms will be arranged such that the second prism is angled at 45°relative to the first prism. For quartz, the angular displacement rangesfrom 30° to 50°.

[0030] Another aspect of the current invention comprises a method ofilluminating a sample comprising: (a) steering two or more excitationlines onto a first surface of a first prism; (b) steering two or moreexcitation lines from the second surface of said first prism to a firstsurface of a second prism; wherein said second prism is angled about 45°from said first prism; (c) steering said two or more excitation linesonto a sample after exiting second surface of said second prism, whereinsaid two or more excitation lines are substantially colinear and/orsubstantially coaxial after exiting said second prism.

[0031] Yet another aspect of the current invention comprises a method ofcontrolling a sequence of excitation lines comprising: (a) obtaining aTTL circuit comprising an electronic stepper wherein said circuit isoperationally connected to one or more lasers having two or moreexcitation lines; (b) and controlling the sequential firing of the oneor more lasers having two or more excitation lines with a clock pulsefrom the circuit, wherein the frequency of firing one laser isequivalent to the frequency of firing a second laser, but phased shiftedso that one or more lasers having two or more excitation lines can besequentially pulsed. The cycle time of one clock pulse may be from 1μsecond to 5 seconds, or from 100 μsecond to 1 second. The length oftime a first laser produces an excitation line may be similar to thelength of time a second laser produces an excitation line. As usedherein, similar means within 20%, within 10%, or more preferably within5% of the time length. Between 2-to-16, or 2-to-8 excitation lines aresequentially pulsed.

[0032] Yet another method of the current invention comprises a method ofcontrolling a sequence of excitation lines comprising: (a) obtaining aTTL circuit comprising an electronic stepper wherein said circuit isoperationally connected to one or more lasers having two or moreexcitation lines; (b) and controlling the sequential firing of the oneor more lasers having two or more excitation lines with a clock pulsefrom the circuit, wherein the frequency of firing a first laser isdifferent from the frequency of firing a second laser. This method maybe used to control 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16lasers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0034]FIG. 1. An example of a PME device where each laser is regulatedindividually by its own power supply (A). The TTL stepper or clock chipcircuit (FIG. 3) chooses which power supply is turned on at a specifictime as it cycles through the five lasers. The beam steering mirrors (B)allow various degrees of adjustment to align the excitation lines sothat once they go through a dual prism assembly (FIG. 2) (C), they willbecome colinear and/or coaxial. The beams enter the dark box (D) wherescattered light is reduced by the use of irises and a long cell. Thedyes are detected by the photomultiplier tube (B) through a collectinglens. The signals are recorded by the oscilloscope (F) where digitalpictures can be analyzed.

[0035]FIG. 2. Inversion dispersion scheme using a dual prism assembly tocombine pulsed multiline excitation laser sources from discretelocations. The beams all enter from the left hitting the prism atvarying angles and positions on the left side of the first prism (upperleft). As they hit the first prism, the laser beams all bendapproximately at a 45-degree angle. At this point the beams are not yetcolinear and/or coaxial but they are spatially closer together thanbefore. As they hit the second prism, they once again hit at varyingangles and positions, and become colinear and/or coaxial as they exitthe prism.

[0036]FIG. 3. TTL Circuit Clock Chip (74174). MR, when low, resets thechip and sets all outputs to low; CP is the Clock Pulse Input. Q0through Q7 are outputs that connect to and signal each of up to eightlasers to fire in sequence.

[0037]FIG. 4A, FIG. 4B and FIG. 4C. Photographic data from theoscilloscope output. Two channels from the oscilloscope were set torecord the clock signal for firing the red laser (top line) and the PMTdetector output (bottom line). Arrows correspond to the red and greenlaser pulses. Data on dilute aqueous solutions containing both BODIPY523/547 and BODIPY 630/650 dyes (FIG. 4A), BODIPY 630/650 dye only (FIG.4B), or BODIPY 523/547 dye only (FIG. 4C) were collected.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0038] I. The Present Invention

[0039] The present invention describes a novel device and approach tofluorescence detection, which has general application for geneticanalysis methodologies with particular emphasis on DNA sequencingtechnologies and high-throughput identification of single nucleotidepolymorphisms (SNPs). The PME technology has two main advantages thatsignificantly increase fluorescence sensitivity: optimal excitation ofall fluorophores in the genomic assay and “color-blind” detection, whichcollects considerably more light than traditional dispersive detectors.The fluorescence detector can be designed to miniaturize DNA sequencingtechnology with a sensitivity enabling direct detection of fluorescentDNA assays from genomic DNA material. The PME is useful for clinicaldiagnostic, forensic, and general sequencing.

[0040] II. Pulsed Multi-line Excitation (PME) Detection

[0041] In the current invention, spectral dispersion or wavelengthdiscrimination of fluorescent dyes is eliminated, which increases theamount of fluorescent signal detected. The sequential pulsed-laserexcitation system using multiple lasers emitting specific wavelengths oflight, which are matched for efficient excitation of a given set offluorophores, can determine selectivity and sensitivity. By matching theabsorption maximum of each fluorophore, the PME technology excites eachdye with the highest quantum efficiency, thus considerably reducing therequired sample size (i.e., the number of fluorescent molecules requiredfor detection). At first glance, replacing one laser with four lasersmay appear counterintuitive to miniaturization. New solid state lasers,however, such as diode pumped Nd:YAG sources or diode lasers are muchsmaller (ca. 2″ long) than standard argon ion lasers and are much moreefficient requiring smaller power supplies for operation. For example,the footprint of four solid-state lasers together is approximately20-fold smaller than a single argon-ion laser system. Simply replacingthe argon-ion laser, which for a DNA sequencer relies on two excitationlines at 488 nm and 514.5 nm, with a equal power 532 nm Nd:YAG canreduce the laser size, but would reduce the excitation/emissionintensities of shorter wavelength dyes, and still would not efficientlyexcite longer-wavelength dyes that have absorption maxima far from thelaser wavelengths.

[0042] For the PME technology to discriminate four fluorescent dyes,four excitation lines are combined by inverse dispersion, which isillustrated but not limited to using a prism assembly or diffractiongrating. The resultant beam would look on average like a “white light”laser beam. However, the solid-state lasers are electronicallycontrolled and are pulsed or fired sequentially as discrete packages ofwavelength specific light. Alternatively, laser sources can be pulsed orfired using a synchronized shutter system. Each dye brightly fluoresceswhen its matched laser source is turned on, while it responds onlyweakly, if at all, to the other three laser pulses. The fluorescencefrom each excitation event is collected using a non-dispersed or “colorblind” method of detection. A non-dispersive detector is a detector inwhich the incident radiation is not separated based on the emissionfluorescence wavelength of different fluorescent dyes. Thus, DNAsequence is determined by the PME technology based on the timecorrelation of detector response to specific wavelengths of excitationlight, and not spectral resolution of emission wavelengths. Switchingthe solid-state lasers on a millisecond timescale is straightforward,hence thousands of 4-laser excitation cycles may be completed in thetime scale for eluting a single base of DNA by capillaryelectrophoresis.

[0043] Moreover, an advantage of the non-dispersed system is that thedetector (i.e., CCD) collects significantly more light, since thefluorescent light is directly coupled to the detector. Typically for thecurrent DNA sequencer, a dispersive element requires highly collimatedlight for effective wavelength separation. Moving the collection lenscloser to the sample can increase the collected fluorescent light, butcollimation is lessened, and spectral selectivity is reduced. Similarly,reducing the distance between the dispersing element and the detectorresults in reduced spectral selectivity. For the non-dispersed system,however, moving the collection lens much closer to the sample or to thedetector increases the collected light, inverse to the square of thedistance, but without sacrificing the selectivity that is provided byfour laser cycling. Thus, the miniaturization process inherentlydelivers more fluorescent light to the detector.

[0044] Typically, miniaturizing a system incurs inevitable penalties insensitivity and selectivity. For example, fluorescent signal is lost asthe laser source becomes smaller in size and power, and selectivity iscompromised because spectral dispersing elements need physical space toseparate emission wavelengths and compressing the spectrometer portionof the detection sacrifices spectral resolution. Consequently, thesample size is increased to offset these losses, which tends tomarginalize the benefits derived by shrinking the conventionaldispersive optical system. The design described herein minimizes thelosses in downsizing instrumentation, but increases the sensitivityconsiderably by the process of miniaturization. The current inventioncomprises a novel detection system that allows the optical components toact synergistically when miniaturized.

[0045] An additional advantage of non-dispersed detection is enhancedsignal-to-noise compared to the current 96-capillary DNA sequencer. Toobtain the wavelength spectrum for each DNA sequence reaction, a largenumber of pixels are read out, and this electronic readout process addsnoise for every pixel read. For the non-dispersed system, all pixelsthat receive light from a particular capillary are “binned” and read outas a single unit, considerably reducing the associated electronic noise.

[0046] III. Fluorophores

[0047] An advantage of using PME or any time-based detection as opposedto wavelength discriminating detection is the increase in the number offluorophores, which can be used. At any given excitation wavelength,there are often only about two or three commercially available dyes thatemit with narrow enough emission bands with sufficiently separatedwavelength maxima that can be individually measured simultaneously (U.S.Pat. No. 6,139,800). If three or more fluorophores can be found, thereis still substantial cross-talk or overlap of the emission spectra thatwill require substantial deconvolution of the spectra with acorresponding increase in the likelihood of error in identifying thespecies.

[0048] One solution to this problem has been the addition of a secondlaser to allow for the simultaneous or sequential detection of up toapproximately six dyes (U.S. Pat. No. 6,139,800). However, this solutionstill has the problem of substantial overlap in the spectra and the needfor signal intensities great enough to be detected after spectraldispersion of the signal.

[0049] Optimally, the way to obtain the highest emission signal possibleis to optically match an excitation source with the absorption maxima ofa dye with a high molar extinction coefficient. This is done for everyfluorophore. However, the excitation source need not match theabsorption maxima exactly, instead, it is important to obtain laser-dyecombinations where each dye has an absorption maxima which substantiallycorresponds with one source wavelength with concomitant emission,coupled with minimal absorption/emission (cross-talk) from thenon-matched laser sources used in the assay.

[0050] A system with four fluorophores used to detect the 4 DNA bases ispreferred. However, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or16 different fluorophores may be used with the PME system.

[0051] A non-limiting list of dyes that may be used in the currentinvention include BODIPY dyes (BODIPY 630/650, BODIPY 650/665, BODIPY589/616 or BODIPY-TR, BODIPY 581/591 BODIPY 523/547 or BODIPY-R6G,5,7-dimethyl-BODIPY (503/512) or BODIPY-FL, 1,3,5,7-tetramethyl-BODIPY(495/503), BODIPY-TMR-X or BODIPY (564/570)-X, BODIPY-TR-X or BODIPY(589/616)-X, BODIPY (530/550), BODIPY (564/570), and BODIPY (558/568)),a zanthene dye, a rhodamine dye (rhodamine green, rhodamine red,tetraethylrhodamine, 5-carboxy rhodamine 6G (R6G), 6-carboxy R6G,tetramethylrhodamine (TMR), 5-carboxy TMR or 5-TAMRA, 6-carboxy TMR or6-TAMRA, rhodamine B, X-rhodamine (ROX), 5-carboxy ROX, 6-carboxy ROX,lissamine rhodamine B, and Texas Red), a fluorescein dye (FITC,5-carboxy fluorescein, 6-carboxy fluorescein, fluorescein diacetate,naphthofluorescein, HEX, TET, 5-carboxy JOE, 6-carboxy JOE, Oregon Green488, Oregon Green 500, Oregon Green 514, erythrosin, eosin), a coumarindye (7-hydroxycoumarin, 7-dimethylaminocoumarin, 7-methoxycoumarin,7-amino-4-methylcoumarin-3-acetic acid or (AMCA), and Pacific Blue), acyanine (Cy) dye (Cy3, Cy3.5, Cy5, Cy5.5, Cy7), a phthalocyanine dye, aphycobiliprotein dye, (B-phycoerythrin (B-PE), R-phycoerythrin (R-PE),and allophycocyanin (APC)), a pyrene and a sulfonated pyrene, (cascadeblue), a squaraine dye, an Alexa dye (Alexa 350, Alexa 430, Alexa 488,Alexa 532, Alexa 546, Alexa 568, Alexa 594) and Lucifer yellow.

[0052] IV. Excitation Sources

[0053] A central principle of the PME technology is the discriminationof a mixture of different fluorophores by the time correlation of“colorblind” fluorescence emission triggered by serially pulsingdifferent excitation lasers. This approach significantly contrasts thatof the widely used method of wavelength discrimination of fluorescenceemission, where a single excitation source, typically an argon ion laser(488 nm and 514.5 nm) excites four spectrally resolvable fluorescentdyes. The dye of this set, which emits at the longest emittingwavelength is usually the least optimally excited, which is due to poorspectral overlap between the excitation source and the dye's absorptionmaximum. This inefficient excitation has been partially overcome by theuse of fluorescence resonance energy transfer (FRET) dye-primers (Ju etal., 1995; Metzker et al., 1996) and dye-terminators (Rosenblum et al,1997) to increase signal intensities. Obviously, the optimal method inobtaining the highest emission signal possible would be matching theexcitation source with the absorption maxima for every fluorophore inDNA sequencing assays.

[0054] The invention may use at least one laser and is flexible toaccommodate as many different lasers as is feasibly possible. There maybe 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more lasers,depending on the system.

[0055] A single laser may produce 1, 2, 3, 4, 5, 6, 7, 8 or moredifferent wavelengths for the excitation of fluorophores with differentabsorption maxima. This can be accomplished using the technique ofStimulated Raman Shifting (SRS). This technique may be employed forconversion to either shorter or longer wavelength(s). The Raman effectenables a laser frequency to be modified by discrete increments, (theStokes and Anti-Stokes shifts). Frequency conversion is accomplished bypassing laser light through a suitable crystal or a stainless steel cellcontaining gas at an elevated pressure, (i.e. several atmospheres).Conversion efficiency for the principal Stokes shift to longerwavelength can be as high as 35%. The nature of the crystal or gasdetermines the frequency output, for example, N₂, O₂, H₂, D₂, and CH₄give shifts of 2330 cm⁻¹, 1550 cm⁻¹, 4155 cm⁻¹, 2987 cm⁻¹, and 2917 cm⁻¹respectively, while Ba(NO₃)₂ gives a shift of 1047 cm⁻¹. A preferredRaman medium for this invention is molecular nitrogen, as 2330 cm⁻¹ isabout the desired spacing between excitation frequencies.

[0056] Until recently, gas lasers have been widely used for theexcitation of “blue” and “green” fluorophores with absorption maximaranging between 488 nm and 543 nm for DNA sequencing applications. Ingeneral, these lasers include the argon ion, the krypton ion, and thehelium-neon (He—Ne) lasers. These lasers are large in size, highlyinefficient and relatively expensive devices. Moreover, the lifetime ofgas laser is approximately 1,000-to-3,000 hours of use, which imposeshigh maintenance cost for these instruments. Despite thesedisadvantages, the argon ion laser has been widely used in automated DNAsequencing instrumentation for 15 years now (Smith et al., 1986; Probeet al., 1987) and is frequently described as the excitation source inmany capillary electrophoresis systems, see below. accomplished byspatially combining the different laser beams into one overlapping“white” beam.

[0057] Other groups have developed devices to combine two or more laserbeams. Conemac (U.S. Pat. No. 6,226,126) describes a laser beam mixerhaving a beam combining element with a transmissive portion and areflective portion. However, this technology requires that the crosssectional shape of the second, third and so forth laser beams bedistorted. Another limitation is that for each laser beam added, thecombined beam must pass through an additional optical element, whichintroduces loss into the system.

[0058] U.S. Pat. No. 5,991,082 discloses a lens system that forms narrowsuperimposable focal lines from multiple focal lines. This system uses aprism with multiple longitudinally arranged facets bounded by parallelridge lines and can be used to obtain a high energy radiation beam foruse in pumping an X-ray laser.

[0059] U.S. Pat. No. 6,215,598 discloses an apparatus for concentratinglaser beams, which comprises collimating devices that converge the laserbeams into a laser beam sheet. A digital optics device shapes andconcentrates the laser beam sheet into a narrow overlapping laser beam.

[0060] In the present invention, an inverse dispersion system can beused to steer the light from multiple lasers onto a single sample.Inverse dispersion uses optical dispersion elements such as a prismassembly or a diffraction grating positioned such that light fromdiscrete locations is steered to be substantially colinear and/orsubstantially coaxial upon exiting the system. The term “substantiallycolinear” means that the laser beams or excitation lines diverge fromeach other at angles of less than 5°. The term “substantially coaxial”means that the laser beams or excitation lines diverge from each otherat angles of less than 5°.

[0061] The inverse dispersion system may be configured as shown in FIG.2 in which five excitation lines from discrete locations are combinedinto a single colinear and/or coaxial line.

[0062] High dispersion equilateral prisms constructed from high gradeglass, quartz or silica are used in one preferred embodiment of theinvention. The preferred orientation for the prisms relative to eachother when using high dispersion flint glass prisms is 40°-50°, or morepreferred 45°. The preferred orientation for the prisms relative to eachother when using quartz prisms is 25°-55° or more preferred 30°-50°.This angle allows efficient overlap of the multiple beams by inversedispersion into a single beam. A two prism assembly is preferred,however, a single prism or an assembly with three or more prisms is alsocontemplated. Similarly, a diffraction grating such as a ruled orholographic grating can be used to combine the multiple beams. Multipleexcitation lines can be steered onto a diffraction grating such that thediffraction of the grating causes the beams to combine.

[0063] In addition to being colinear, the beams may be coaxial, with allof the laser beams passing through the same columnar space in thesample. The same sample molecules will be exposed to each of the laserbeams sequentially in turn as the lasers are fired.

[0064] The inverse dispersion approach uses the same principle firstdemonstrated by Sir Isaac Newton, but in reverse direction. In hisexperiment, collimated white light, from the sun, passed through anequilateral prism, and the various wavelengths became separated byangle. The beam of light passes into the prism, forming a non-zero anglewith respect to the normal to the entrance surface. According to Snell'slaw, all of the rays will be bent towards the normal as the light passesinto the more optically dense medium. Due to dispersion of the glass,the shorter wavelengths deviate more. When the rays exit the prism, allare bent a second time, but again, the shorter wavelengths bend more.The result is that the shorter wavelengths now have an angularseparation from the longer wavelengths. Blue light is deviated more thangreen, which is deviated more than yellow, and that in turn more thanred.

[0065] This process may be reversed, and that is the principle utilizedfor inverse dispersion. If the separated rays are made to trace pathsthat are just the reverse of above, then the various wavelengths arecombined into a single beam of “white” light. For example, light fromlasers, light emitting diodes, arc lamps, incandescent lamps, etc. maybe combined (after collimation and spectral filtering, if appropriate)by this inverse dispersion method. The shorter wavelength rays or beamsenter the prism at the appropriate larger off-normal angle than thelonger wavelength beams. When the correct angles are determined fromSnell's law, the beams will all combine into a single coaxial beam. Ifdesired, the beams may be spatially offset to provide colinear beamsthat, while parallel to each other, pass through the sample at slightlydifferent positions. In the former case, the fluorescence from all ofthe beams may be imaged onto a single detector. In the latter case, thebeams may be imaged onto four or more separate detectors, or separateregions of one detector, such as a CCD camera. If the light sources arepulsed in rapid succession, the combined beam appears to be white ornearly so. If the pulsing is slow enough for the eye to follow, thecombined beam will exhibit a changing color pattern originating from thesame spatial location.

[0066] The prism is typically used at the minimum deviation angle,whereby the entering and exiting angles for a given beam are equal, oras nearly so as practical. The apex bisector will also bisect the angleformed by the entering and exiting beam. If the prism is then rotated,then these two angles are no longer equal. This is advantageous in thatit increases both the overall deviation angle and the amount ofdispersion. However, this also causes anamorphic changes in the beamdiameter. Such anamorphic expansion can be useful if it is desirable tochange a round beam cross section to an oblong one, or an oblong beamshape to a round shape. If the inverse dispersion combining of beams isto be done with minimal distortion of the original beam shape, then theminimum deviation angle is preferred.

[0067] The angular separation of the beams is increased in proportion tothe number of prisms the beam passes through. For example, the use oftwo identical prisms doubles the angular separation. The anamorphic beamchanges can be nearly canceled by using a pair of prisms at non-minimumdeviation angles. Use of high dispersion glass, such as flint glass alsoincreases the angular separation of the incoming beams. This in turnreduces the distance needed to achieve spatial separation of theincoming beams, and provides for a more compact optical apparatus. As anexample, the present apparatus utilizes two F2 flint glass prisms.

[0068] A diffraction grating may also be used as a suitable inversedispersion element. For a grating exposed to a collimated beam of whitelight, the beam is diffracted in accordance with the grating equation.If it is a transmission grating, the beam is diffracted away from astraight line path, called the zero order, with the longer wavelengthsdeviating at a larger angle than the shorter wavelengths. The order ofthe dispersed wavelengths is the reverse of that for prisms. If areflection grating is used, again the longer wavelength beam is deviatedfurther from the zero order reflection than a shorter wavelength. Aswith the prism combiner above, beams of different wavelengths incidenton the grating in the reverse direction will provide the same sort ofinverse dispersion, leading to a colinearity of the beams. They may bemade coaxial if the beams are incident on the same area on the gratingbut at the appropriate angles for each of the wavelengths.Conventionally ruled gratings are suitable for this purpose, howeverholographic gratings generally exhibit less scattered light. Gratingsgenerally can be obtained that have considerably higher dispersion thanprisms, and hence dispersion angles are larger and the spacing betweenthe light sources can be reduced. They are generally less efficient, sothat light losses are greater. Gratings and prisms are both sensitive tothe polarization of the light. Since the fluorescence emission is alsosensitive to the direction of the polarization, proper orientation ofthe electric vector of the light should be considered. Polarizationrotation devices may be added to improve the transmission efficiency.

[0069] VI. Collection Devices

[0070] Of the possible choices for detection of fluorescence, theoptimum one will depend upon the level of fluorescence intensity. Forall detectors described herein, a photon striking the detector isconverted into a charge carrier, which is then detected electronically.Charge carriers, however, are generated by extrinsic processes unrelatedto the fluorescence signal from a variety of sources: thermalgeneration, cosmic rays, and natural radioactivity. All carriersgenerated both from fluorescence and from unrelated sources contributeto shot noise, a well-understood statistical phenomenon. Of theextrinsic sources of excess carrier noise, thermal generation ofcarriers is usually the dominant extrinsic noise source, which can bereduced by cooling the detector. Fundamentally all that is needed forsatisfactory detection is that the number of charge carriers generatedby fluorescence during an observation time window is much greater thanthe square root of the number of all carriers generated during the sametime interval. However, once the carriers leave the detector, theamplifying electronics will introduce noise as well, and this electronicnoise may dominate. To avoid this situation, devices have been inventedthat incorporate their own very low noise amplifiers. There are two suchdevices: the photomultiplier tube (PMT) and the silicon avalanchephotodiode (APD). Another approach to reducing amplifier noise is tocollect carriers for a period of time and then rapidly read thecollected charges out. This mode of operation is used for photodiodearray charge-coupled device (CCD) detectors.

[0071] For light levels with high enough signals that the noisegenerated in the external amplifiers is negligible, the simplest devicefor fluorescence detection is the silicon PIN detector. This deviceconsists of a thin hole rich region (p) and an electron rich region (n)separated by thick carrier deficient region (i). It is back-biased witha negative voltage applied to the p side and a positive voltage appliedto the n side. Light striking it penetrates the p region and is absorbedin the i region generating an electron hole pair with the electron beingattracted to the n side and the hole to the p side creating a currentthrough the device. The quantum efficiency of this process is very high(˜80%). Because it is the simplest, most compact, and cheapest detector,the silicon PIN is a preferred detector. Other, more sensitive detectorsmay also be used for detecting low levels of light emission for amulti-capillary electrophoresis system and direct detection assays.

[0072] The silicon PIN detector can be made suitable for the detectionof very low light levels by introducing carrier gain into it. A photonstrikes the detector creating an electron hole pair. The electron isaccelerated by an electric field and creates additional carriers byionization. The additional electrons are accelerated to produce morecarriers resulting in an avalanche process and current gain. Siliconavalanche detectors operate in two modes analogous to that of the PMT:an analog current measurement mode and a digital counting mode. Whenoperated in the analog mode, the current gain (˜300) is less than thatof a PMT (˜10⁵-10⁶), but with exception to the lowest light levels, thesignal is still much larger than the noise in the external amplificationcircuit. In addition, the wavelength response range (300 to 1100 nm) ofsilicon detectors is much wider than any individual PMT photocathode,covering the fluorescent maximum of any dye that might be used for DNAsequencing instrumentation.

[0073] Alternatively, a silicon APD can be thermoelectrically cooled andoperated in the Geiger counting mode, where individual fluorescencephotons are counted. This provides a high quantum efficiency (˜80%) withdark count levels approaching a cooled PMT (quantum efficiency typically10%). The thermoelectrically cooled silicon APD provides a compact formcombined with state-of-the-art sensitivity. While higher sensitivity maynot be required for detecting fluorescent signals from standardsequencing reactions, silicon APDs in the counting mode can be ideal fordetecting fluorescent signals directly from genomic DNA assays (i.e.,without amplification).

[0074] Detectors with the required characteristics are commerciallyavailable and include the simple silicon PIN detector, the silicon APD,or a photodiode array (CCD). The simple silicon detector is thecheapest, the silicon avalanche is the most sensitive, and the CCD ismost useful for multiple capillary systems as it provides many detectorsin a single unit.

[0075] VII. Coupled Systems

[0076] The PME technology has sufficient flexibility for coupling to avariety of formats, for example, the PME system can be coupled toconventional capillary electrophoresis (CE) or to separation and/orpurification using high-density arrays/biochips. Ideally, a DNAsequencing system capable of direct detection of fluorescent assays forgenomic DNA should (i) optimally excite all fluorescent dyes, (ii) becapable of efficiently collecting photons over a large part of the UV,visible and infrared spectrum, (iii) continuous monitoring offluorescent signals in high-throughput array or high-density formats,(iv) maximize fluorescence emission signals for detection, (v) beconfigured to minimize background scattered light, and be automatedusing replaceable gel matrices.

[0077] a. Capillary Electrophoresis

[0078] The PME fluorescence detection system of the present inventioncan be coupled to conventional capillary electrophoresis (CE) as apreferred method for resolving DNA fragments.

[0079] Microcapillary array electrophoresis generally involves the useof a thin capillary or channel, which may or may not be filled with aparticular separation medium. Electrophoresis of a sample through thecapillary provides a size-based separation profile for the sample. Theuse of microcapillary electrophoresis in size separation of nucleicacids has been reported in, e.g., Woolley and Mathies (1994). The highsurface to volume ratio of these capillaries allows for the applicationof higher electric fields across the capillary without substantialthermal variation across the capillary, consequently allowing for morerapid separations. Furthermore, when combined with confocal imagingmethods, these methods provide sensitivity in the range of attomoles,which is comparable to the sensitivity of radioactive sequencingmethods. Microfabrication of microfluidic devices includingmicrocapillary electrophoretic devices has been discussed previously(e.g., Jacobsen et al., 1994; Effenhauser et al., 1994; Harrison et al.,1993; Effenhauser et al., 1993; Manz et al., 1992; and U.S. Pat. No.5,904,824). Typically, these methods comprise photolithographic etchingof micron scale channels on a silica, silicon or other crystallinesubstrate or chip, and can be readily adapted for use in the presentinvention. In some embodiments, the capillary arrays may be fabricatedfrom the same polymeric materials described for the fabrication of thebody of the device, using the injection molding techniques describedherein.

[0080] Tsuda et al., (1990), describes rectangular capillaries, analternative to the cylindrical capillary glass tubes. Some advantages ofthese systems are their efficient heat dissipation due to the largeheight-to-width ratio and, hence, their high surface-to-volume ratio andtheir high detection sensitivity for optical on-column detection modes.These flat separation channels have the ability to performtwo-dimensional separations, with one force being applied across theseparation channel, and with the sample zones detected by the use of amulti-channel array detector.

[0081] In many capillary electrophoresis methods, the capillaries, e.g.,fused silica capillaries or channels etched, machined or molded intoplanar substrates, are filled with an appropriate separation/sievingmatrix. Typically, a variety of sieving matrices are known in the art,which may be used in the microcapillary arrays. Examples of suchmatrices include, e.g., hydroxyethyl cellulose, polyacrylamide, agaroseand the like. Generally, the specific gel matrix, running buffers andrunning conditions are selected to maximize the separationcharacteristics of the particular application, e.g., the size of thenucleic acid fragments, the required resolution, and the presence ofnative or undenatured nucleic acid molecules. For example, runningbuffers may include denaturants, chaotropic agents such as urea or thelike, to denature nucleic acids in the sample.

[0082] The use of replaceable gel matrices, which suppresselectroendoosmotic flow and DNA-capillary wall interactions such aspolydimethylacrylamide (Madabhushi, 1998), may be used forelectrophoretic separations in the present invention.

[0083] b. Chromatographic Techniques

[0084] Alternatively, chromatographic techniques may be coupled to thePME fluorescence detection system of the present invention. There aremany kinds of chromatography, which may be used including liquidchromatography, HPLC and many specialized techniques, such as reversephase HPLC, normal phase HPLC, anion exchange, cation exchange,denaturing HPLC, size exclusion or gel permeation, and hydrophobicinteraction.

[0085] C. Microfluidic Techniques

[0086] Microfluidic techniques can be used for fluid flow with the PMEsystem, and includes the use of a platform such as microcapillaries,designed by ACLARA BioSciences Inc., or the LabChip™ “liquid integratedcircuits” made by Caliper Technologies Inc. Miniaturizing some of theprocesses involved in genetic analysis has been achieved usingmicrofluidic devices. For example, published PCT Application No. WO94/05414, by Northrup and White, incorporated herein by reference,reports an integrated micro-PCR™ apparatus for collection andamplification of nucleic acids from a specimen. U.S. Pat. No. 5,304,487to Wilding et al., and U.S. Pat. No. 5,296,375 to Kricka et al., discussdevices for collection of cell containing samples and are incorporatedherein by reference. U.S. Pat. No. 5,856,174 describes an apparatus,which combines the various processing and analytical operations involvedin nucleic acid analysis and is incorporated herein by reference.

[0087] d. Chip Technologies

[0088] Specifically contemplated by the present inventors for combiningwith the PME system are chip-based DNA technologies. These techniquesinvolve quantitative methods for analyzing large numbers of genesrapidly and accurately.

[0089] Chip based technologies that can be used in the current inventioninclude the those described in U.S. Pat. No. 6,153,379 and Shumaker etal. (1996) where a method of analyzing oligonucleotides is described inwhich oligonucleotides are extended with fluorescent dideoxynucleotides,and detected using an automated fluorescent DNA sequencer. Theoligonucleotide length identifies the known mutation site, and thefluorescence emission of the ddNTP identifies the mutation. Anothermethod of analyzing oligonucleotides involves using template DNAannealed to an oligonucleotide array. The analysis is done using aPhosphor Imager and alpha-³²P labels. Kurg et al., (2000) describes anintegrated system with DNA chip and template preparation, multiplexprimer extension on the array, fluorescence imaging, and data analysis.The method includes annealing DNA to immobilized primers, which promotesites for template-dependent DNA polymerase extension reactions usingfour unique fluorescently labeled dideoxy nucleotides. A mutation isdetected by a change in the color code of the primer sites.

[0090] Motorola BioChip Systems has the I-based SNP systems with arraytechnology centered on a three-dimensional gel pad format consisting offlexible content architectures.

[0091] The MassARRAY system, developed by SEQUENOM (U.S. Pat. Nos.5,547,835, 6,238,871, and 6,235,478) has a platform capable of highthroughput SNP analysis using enzymology, bioinformatics andminiaturized chip-based disposables with mass spectrometry detection.The MassARRAY technology can be used to distinguish genotypes usingMALDI-TOF mass spectrometry. DNA fragments associated with geneticvariants are simultaneously separated and detected, measuring target DNAassociated with SNPs and other forms of genetic variation directly.

[0092] f. Bead Technologies

[0093] The PME system may be coupled with microbeads containing boundDNA or RNA segments. These beads may be plain or coated with materialsuch as biotin, terminal amines, or Protein G to facilitate binding ofthe biomolecule to the bead. Microbeads may be used in conjunction with,for example, microfluidic systems or electrophoretic systems and PMEdetection. The beads may be porous or solid and made of polymers such aspolystyrene or agarose and may optionally contain magnetic particles,such as those obtained from Dynal thus allowing use in magneticseparation techniques. The beads may also be porous thereby providingincreased surface area for binding. Magnetic beads are used in a mannersimilar to polymer beads. However, these beads contain a magnetic sourcesuch as Fe₂O₃ or Fe₃O₂ that can be used for rapid and simple separation.

[0094] VIII. Continuous Fluorescence Monitoring

[0095] To capture minute fluorescent signals for multiple capillaryarray or other formats derived from direct assays, the high duty cycleof continuous monitoring systems has significant advantage over scanningsystems. Moreover, continuous systems have other benefits that simplifythe mechanical operation of the system, such as no moving mechanicalstages that wear down, break down, or become misaligned. These systemsuse the laser light power more efficiently allowing greater operation offluorescence excitation under photobleaching conditions. Basically,there are two known methods for continuous monitoring of fluorescentsignals, namely on-column and post-column detection. Both methods can beused with the PME technology of the current invention for DNA sequencingapplications using capillary electrophoresis.

[0096] a. On-column detection

[0097] The first on-column detection schemes were described using singlecapillary systems (Luckey et al., 1990; Swerdlow et al., 1990; Drossmanet al., 1990; Cohen et al., 1990). In 1990, the Smith group describedthe first 4-color on-column system using a multi-line argon-ion laser(488 nm and 514.5 nm) to illuminate a single capillary. The fluorescenceemitted from FAM, JOE, TAMRA, and ROX dye-labeled sequencing reactionswas collected orthogonal to the excitation source and using a set ofbeamsplitters, the emitted fluorescence was directed to a set of 4 PMTdetectors (Luckey et al., 1990). Karger et al. described the firston-column, spectral dispersion system using a CCD camera (Sweedler etal., 1991) to detect emission wavelengths approximately in the range of500 nm to 650 nm derived from the fluorescence of a 4-color sequencingreaction. Similar to the Smith design, the fluorescence was collectedperpendicular to the excitation path, but the authors describe theunique feature of 4 target areas that correspond to the emissionproperties of FAM, JOE, TAMRA, and ROX dye-labeled reactions and thebinning of pixels within the target areas for enhanced readout speed andreduced readout noise (Karger et al., 1991).

[0098] The first capillary array instrument was a modified DuPontGENESIS 2000 DNA sequencing instrument (Probe et al., 1987), which theslab gel was replaced with a 12-capillary array (Zagursky et al., 1990).An argon-ion laser beam (single line 488 nm) was scanned across thecapillaries and fluorescence was detected using the 510 nm and 540 nmresolved, two PMT detector scheme. The Mathies group has described aconfocal-fluorescence system, which has potential for improved signal tonoise and scanned back and forth using a motor-driven translation stageacross a 24-capillary array (Huang et al., 1992). Fluorescence wascollected using a 180° geometry to the argon ion laser (488 nm)excitation source by confocal detection. Originally, a two PMT detectorsystem coupled to a two-dye binary coding method was used with differentmole fraction combinations of FAM and JOE dye reactions to differentiatethe four-termination reaction set (Huang et al., 1992). Recently, theMathies group developed a 4-color confocal scanning detection systemthat directs the fluorescence emission through a number of differentdichroic beamsplitters to 4 PMT detectors (Kheterpal et al., 1996). The4-color confocal scanning system described here represents the coretechnology for the commercially available 96-capillary MegaBACE 1000instrument (Molecular Dynamics). More recently, the Riken group hasdescribed a 384-capillary DNA sequencer, which uses an argon ion laserin a scanning mode and splits the fluorescence emission signal to 4different band-pass filters coupled to dedicated PMT detectors (Shibataet al., 2000). Although mechanical scanning systems can uniformlyilluminate each capillary in the array, in general, they can beproblematic due to breakdown and misalignment, low duty cycle, andpotential photobleaching by duty cycle compensation with higher laserpower levels.

[0099] Several side-entry illumination schemes directly throughcapillary arrays have been problematic in scaling from a singlecapillary to array systems, mainly because of reflection and refractionof the laser beam at the capillary boundaries. The Yeung group describeda 10-capillary system that used axial beam illumination and CCDdetection, in which individual optical fibers coupled via an argon ionlaser were directly inserted into the ends of the capillary tubes(Taylor et al., 1993). The intrusion of optical fibers into separatecapillaries, however, affected the electroendoosmotic flow and increasedthe possibility for contamination and clogging (Lu et al., 1995). Thisgroup also described illumination by a line of laser light focusedacross the array using a plano-convex cylindrical lens to illuminate a100-capillary array (Ueno et al, 1994); however, this designinefficiently used less than 0.5% of the laser power to illuminate eachcapillary (Lu et al., 1995). Quesada and Zhang (1996) described an8-capillary prototype in which argon ion illumination and fluorescencedetection were achieved by using individual optical fibers constructedin an orthogonal geometry and imaged through a spectrograph using a CCDcamera. Scaling beyond the initial individual optical fiber design,however, was reported to be problematic because of the increased demandon laser power, and the bulkiness and irregular alignment of the opticaljunction connectors to each capillary tube (Quesada et al., 1998).

[0100] To address the loss of laser illumination by refraction, twoindependent groups have demonstrated that refracted laser light at thecapillary surface can be focused repeatedly under optimized opticalconditions and produce a waveguiding effect (Quesada et al., 1998;Anazawa et al., 1996). Quesada et al. (1998) demonstrated that abi-directionally illuminated waveguide system using an argon ion lasershowed good illumination across a 12-capillary array with a differenceof less than 10% across the array, and with potential for scaling to a96-capillary system with near uniform illumination. Alternatively, indexmatching of the capillary array has also been shown to reduce laserlight refraction and scattering across the array (Lu et al., 1995), butto a smaller degree than waveguide illumination (Quesada et al., 1998).

[0101] b. Post-column detection

[0102] The first post-column detection schemes also described singlecapillary systems (Swerdlow et al., 1990; Swerdlow et al., 1991). TheDovichi group first reported a 4-color post-column detection systemusing a sheath flow cuvette (Swerdlow et al., 1990). These authorsdemonstrated the sheath flow concept using the four-spectral channelsystem based on the work described by the Smith group (Luckey et al.,1990) and a two-spectral channel system based on the work described byProber et al. (1987). Unlike the beamsplitter design, the four-spectralchannels were discriminated using a rotating wheel containing 4 specificband-pass filters, which was synchronized to a sector wheel thatalternated the excitation source between an argon-ion laser (488 nm) anda green He—Ne laser (543.5 nm). The appropriate orientation of thefilter wheel directed the specific emission light wavelengths of FAM,JOE, TAMRA, and ROX dye-labeled sequencing reactions to a single PMTdetector. The two-spectral channel, two intensity system used a singleargon-ion laser (488 nm) to excite the 4-different succinyl-fluoresceindye-labeled reactions, which have limited spectral resolution. Theemission fluorescence centered at 510 nm and 540 nm was uniquely splitusing a single dichroic mirror and detected with two PMT detectors. Theassignment of the nucleotide sequence was performed by determining theratio of baseline-corrected peak intensities (Prober et al., 1987).

[0103] The post-column sheath-flow approach has the advantage ofeliminating excitation light scattering at the capillary surfaces and inilluminating all capillary tracks simultaneously. Kambara and Takahashidescribed the first multiple sheath-flow capillary array system using aHe—Ne laser (594 nm) and single color Texas Red (ROX) labeled DNAsequencing reactions (Kambara et al., 1993). This system was laterdeveloped into a 4-color system using the combined excitation lines fromboth an argon ion laser (488 nm) and a YAG laser (532 nm) tosimultaneously irradiate FAM, JOE, TAMRA, and ROX dye-labeled reactions.The fluorescence was dispersed using an image-splitting prism, passedthrough 4 different optical filters, and detected as two-dimensionalline images using a cooled CCD camera (Takahashi et al., 1994). TheHitachi technology described here represents the core technology for thecommercially available 96-capillary 3700 DNA sequencer instrument(Applied Biosystems).

[0104] Another application of the sheath flow approach to post-columndetection was described in U.S. Pat. No. 6,139,800 where fluorescentdetection of labeled particles is accomplished for capillaryelectrophoresis. Multiple wavelength sources excite the labeledparticles and multiple wavelength discriminating detectors detect thesample emissions.

[0105] Capillary array sheath-flow cuvettes require careful attention tohydrodynamic focusing, which can be achieved by uniformly spacing thecapillaries in the cuvette holder. Recently, the Dovichi group hasdescribed two sheath flow cuvettes, the rectangular cuvette that istapered to force 5-capillaries to squeeze together (Zhang et al., 1999)and the micro-machined cuvette with uniformly spaced etched grooves toalign 16 individual capillaries (Crabtree et al., 2000). Of the twodesigns, the latter one shows more promise for scaling to a 96-capillaryarray.

[0106] IX. Obtaining High Sensitivity and Low Background Scattered Light

[0107] In 1990, several groups reported limit of detection valuescorresponding to 10⁻¹⁹ moles for CE systems and were performed using a10⁻¹¹ M solution of fluorescein flowing continuously in an opencapillary (Swerdlow et al., 1990; Drossman et al., 1990). These systems,however, were roughly 10-fold less sensitive than the sheath flowdetector system, which has a reported detection limit of 10⁻²⁰ moles(Swerdlow et al., 1990; Kambara et al., 1993). Coupled to an APDoperating in the Geiger counting mode, this sheath flow system describedrecently by the Dovichi group showed a limit of detection of 130±30fluorescein molecules (Zhang et al., 1999).

[0108] The number of fluorescence counts generated is the product of twofactors: (i) the number of fluorescence photons generated and (ii) theoverall counting efficiency. The number of fluorescence photonsgenerated is given by${N\quad p} = {\frac{\sigma \quad N\quad P}{A\quad h\quad \nu}Q\quad Y\quad t}$

[0109] where N is the number of dye molecules being excited, P is thelaser power (J/s), σ is the absorption cross-section (cm²), A is thearea of the laser beam, hv is the energy of an excitation photon (J), QYis the quantum yield for fluorescence, and t is the observation time(s). The overall counting efficiency is given by${Eff} = \frac{S\quad {A \cdot Q}\quad E}{4\pi}$

[0110] where SA is the solid angle of fluorescent light collection (insteradians) and QE is the quantum efficiency of the detector. Assumingthat the cross-section is 3.8×10⁻¹⁶ cm² (ε=100,000 liters/(mol-cm)), thewavelength of the excitation is 600 nm, the QY is unity, the numericalaperture is unity, and the quantum efficiency is 0.8, then${N\quad p} = {73\frac{NPt}{A}}$

[0111] The PME system of the current invention is useful as a DNAsequencing device for analyzing SNPs directly from genomic DNA withoutcloning or PCR amplification. Estimating 10⁶ white blood cells per cm³of blood, one calculates 73,000 counts in a second could be expected fora single SNP probing of 1 ml of blood without any concentration ofsolution with a laser power of 1 mW and an area of 1 cm². Concentrationwould reduce A without reducing N. Thus, there is an easily detectablesignal without electrophoresis or heroic measures, granted that thesequencing assays are free of unincorporated dye. Note that focusing thelaser reduces N, but simultaneously reduces A by the same factor so thatthe signal does not depend upon focusing as long as the numericalaperture can be maintained (defocusing limit) or the dye is notdestroyed by two photon absorption effects (tight focusing limit).

[0112] The situation is somewhat different when determining a number ofSNPs simultaneously. Then electrophoresis becomes necessary in order toseparate the various fragments. Typically in Sanger sequencing, a10-to-30 μL sample is introduced into the 3700 DNA sequencinginstrument, but only about 10 μL is actually introduced into thecapillary. This is a loss in N of about a factor of 1000-to-3000reducing N to ˜100-to -300. However, if using the sheath flow approach(Anazawa et al., 1996; Zhang et al., 1999), the dye-tagged fragmentsemerging from a 50 μm ID capillary will occupy a cylinder about 2 mmlong and perhaps 100 μm in diameter. Its cross-sectional area A will be2×10⁻³ cm² and the number of counts in a 1 s counting interval will be${N\quad p} = {{73\frac{300 \cdot 0.001 \cdot 1}{0.002}} = {11,000}}$

[0113] This calculation is consistent with the report by Zhang et al.(1999) that 130 fluorescein molecules emerging from a 50 μm capillarycould be detected in 0.2 sec counting time. It will be possible toreduce the wastage factor of 3000 cited above to perhaps 100 by devisinglow volume methodologies to use a 1 μL sample.

[0114] Thus, a capillary electrophoresis system that utilizes multiple,compact solid-state lasers and laser diodes coupled to highly efficientdetection devices, which employ continuous illumination and sheath flowdetection features is a preferred embodiment of the current invention.These integrated technologies are well suited for the application of thePME technology and have sufficient feasibility for direct detectionassays.

[0115] X. Single Nucleotide Polymorphisms (SNPs)

[0116] Spontaneous mutations that arise during the course of evolutionin the genomes of organisms are often not immediately transmittedthroughout all of the members of the species, thereby creatingpolymorphic alleles that co-exist in the species populations. Oftenpolymorphisms are the cause of genetic diseases. Several classes ofpolymorphisms have been identified. For example, variable nucleotidetandem repeats (VNTRs) are polymorphic and arise from spontaneous tandemduplications of di-or trinucleotide repeated motifs of nucleotides. Ifsuch variations alter the lengths of DNA fragments generated byrestriction endonuclease cleavage, the variations are referred to asrestriction fragment length polymorphisms (RFLPs). RFLPs are been widelyused in human and animal genetic analyses and forensic and paternitytesting.

[0117] Another class of polymorphisms is generated by the replacement ofa single nucleotide. Such single nucleotide polymorphisms (SNPs) rarelyresult in changes in a restriction endonuclease site. SNPs are the mostcommon genetic variations and occur once every 300-to-1000 bases, andseveral SNP mutations have been found that affect a single nucleotide ina protein-encoding gene in a manner sufficient to actually cause agenetic disease. SNP diseases are exemplified by hemophilia, sickle-cellanemia, hereditary hemochromatosis, late-onset Alzheimer disease, etc.

[0118] SNPs can be the result of deletions, point mutations andinsertions and in general any single base alteration, whatever thecause, can result in a SNP. The greater frequency of SNPs means thatthey can be more readily identified than the other classes ofpolymorphisms. The greater uniformity of their distribution permits theidentification of SNPs “nearer” to a particular trait of interest. Thecombined effect of these two attributes makes SNPs extremely valuable.For example, if a particular trait reflects a mutation at a particularlocus, then any polymorphism that is linked to the particular locus canbe used to predict the probability that an individual will be exhibitthat trait.

[0119] Several methods have been developed which can be combined withPME technology to screen polymorphisms and some non-limiting examplesare listed below. Such methods include the direct or indirect sequencingof the site, the use of restriction enzymes where the respective allelesof the site create or destroy a restriction site, the use ofallele-specific hybridization probes, the use of antibodies that arespecific for the proteins encoded by the different alleles of thepolymorphism, or any other biochemical interpretation.

[0120] a. DNA Sequencing

[0121] Traditionally, DNA sequencing has been accomplished by the“dideoxy-mediated chain termination method,” also known as the “SangerMethod” (Sanger, F., et al, 1975), which involves the chain terminationof DNA synthesis by the incorporation of 2′, 3′-dideoxynucleotides(ddNTPs) using DNA polymerase. The reaction also includes the natural2′-deoxynucleotides (dNTPs), which extend the DNA chain by DNAsynthesis. Thus, balanced appropriately, competition between chainextension and chain termination results in the generation of a set ofnested DNA fragments, which are uniformly distributed over thousands ofbases and differ in size as base pair increments. Electrophoresis isused to resolve the nested set of DNA fragments by their respectivesize. The fragments are then detected by the previous attachment of fourdifferent fluorophores to the four bases of DNA (i.e., A, C, G, and T),which fluoresce at their respective emission wavelengths afterexcitation at their respective excitation wavelengths using PMEtechnology. The DNA sequencer may be based on an electrophoresis systemwith the throughput capacity of a single column, 4, 8, 16, 48, 96 or384-capillary instrument or may integrate with other separationplatforms, including high-density chip arrays.

[0122] Similar methods which can be used with PME technology include the“chemical degradation method,” also known as the “Maxam-Gilbert method”(Maxam, A. M., et al., 1977). Sequencing in combination with genomicsequence-specific amplification technologies, such as the polymerasechain reaction may be utilized to facilitate the recovery of the desiredgenes (Mullis, K. et al., 1986; European Patent Appln. 50,424; EuropeanPatent Appln. 84,796, European Patent Application 258,017, EuropeanPatent Appln. 237,362; European Patent Appln. 201,184; U.S. Pat. No.4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat. No. 4,683,194).

[0123] b. Primer Extensions Methods for SNP Detection

[0124] A preferred assay for the detection of multiple SNPs is thesingle nucleotide primer extension method, which has also been calledsingle nucleotide incorporation assay and primer-guided nucleotideincorporation assay. These methods rely on the specific hybridization ofan identically complementary oligonucleotide sequence to the geneticregion or target of interest, which has been amplified by the polymerasechain reaction (PCR) or cloned using standard molecular biologytechniques (Sambrook et al., 1989). However, unlike the Sanger reaction,the primer extension method is assayed using either single unlabeled orlabeled dNTPs, 3′-modified-dNTPs, base-modified-dNTPs, oralpha-thio-dNTPs or a mixture of ddNTPs, which all can chain terminateDNA synthesis under appropriate conditions following the incorporationof a single nucleotide (U.S. Pat. Nos. 4,656,127; 5,846,710; 5,888,819;6,004,744; 6,013,431; 6,153,379, herein incorporated by references).Here, the word usage of 2′-deoxynucleoside triphosphate,2′-deoxyribonucleoside triphosphate, dNTP, 2′-deoxynucleotide,2′-deoxyribonucleotide, nucleotide or natural nucleotide are used assynonymous terms and used interchangeably in the current patentdocument.

[0125] 1. Using Single Unlabeled dNTPs

[0126] A method, called pyrosequencing, uses singly added unlabeleddNTPs and is based on a repetitive cyclic method of start-stop DNAsynthesis of single nucleotide addition. Pyrosequencing is amini-sequencing technique and relies on a multi-enzyme cascade togenerate light by luciferase as the mode of detection (Nyren et al.,1993; Ronaghi et al., 1998). PCR amplified DNA fragments, which containa 5′-biotin group are immobilized on streptavidin-coated magnetic beads.An incorporated unlabeled nucleotide event is monitored by the releaseof inorganic pyrophosphate and the subsequent release of light followingthe primer extension step. Because pyrosequencing is a cyclic DNAsequencing strategy, the placement of the oligonucleotide immediatelyadjacent to the 5′-position is not always required, and the primer canbe placed within the sequencing read-length of the method, usually 20bases. Major disadvantages with the pyrosequencing technique are themethod has low sensitivity, the high cost of the reagents, particularlythe enzymes, and sequence difficulties with homopolymer repeats (i.e.,AAAAA) and high “GC” rich regions.

[0127] 2. Using Single Labeled dNTPs

[0128] Unlike the pyrosequencing method, which can extend the primerbeyond a single nucleotide position, other investigators have reportedsingle nucleotide incorporation assays using single nucleotides labeledwith radioactive, non-radioactive, or fluorescent tags (Sokolov, 1990;Syvanen et al., 1990; Kuppuswamy et al., 1991; Prezant and Ghodsian,1992). In these strategies, the placement of the oligonucleotide isimmediately adjacent to the 5′-position of the single nucleotidemutation site under investigation. Sokolov showed the specificincorporation and correct identification of single nucleotide sequencesof a known sequence in the cystic fibrosis gene using alpha ³²P-dCTP andalpha ³²P-dGTP (Sokolov, 1990). In another report, a similar approachwas described for the detection of the Δ508 mutation in the cysticfibrosis gene and point mutations in exon 8 of the factor IX gene(Hemophilia B) (Kuppuswamy et al., 1991). Following PCR amplification ofspecific target regions, specific oligonucleotides, which hybridizedimmediately adjacent to the 5′-position of the mutation underinvestigation, were extended by one nucleotide using single alpha³²P-dNTPs. Moreover, a dual labeling strategy for SNP detection wasreported for exon 4 of the apolipoprotein E gene using differentcombinations of ³H-labeled dTTP, alpha ³²P-labeled dCTP, ordigoxigenin-11-dUTP. Following immobilization of PCR-amplified fragmentson avidin-coated polystyrene beads, single nucleotide extension assayswere performed and incorporated nucleotides were detected using a liquidscintillation counter at different window settings for ³H and ³²Pradioactivity or colorimetrically using an alkaline phosphatase assay(Syvanen et al., 1990). A similar method, called Trapped-OligonucleotideNucleotide Incorporation (TONI) using a biotinylated primer immobilizedon streptavidin magnetic beads and singly added alpha ³²P-labeled dNTPswas described for genetic screening of mitochondrial polymorphisms anddifferent hemoglobin genotypes (Prezant and Ghodsian, 1992).

[0129] 3. Using Single 3′-modified dNTPs

[0130] Metzker et al. proposed the base addition sequencing strategy(BASS), which is a mini-sequencing technique and involves stepwisesingle nucleotide sequencing by repetitive cycles of incorporation of3′-O-modified nucleotides, detection of the incorporated nucleotide, anddeprotection of the 3′-O-modified nucleotide to generate the 3′-OHsubstrate and allow for the next cycle of DNA synthesis (Metzker et al.,1994). Eight different 3′-O-modified dNTPs were synthesized and testedfor incorporation activity by a variety of DNA polymerases.3′-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown tobe incorporated by several thermostable DNA polymerases. Base specifictermination and efficient photolytic removal of the 3′-protecting groupwas demonstrated. Following deprotection, DNA synthesis was reinitiatedby the incorporation of natural nucleotides into DNA. The identificationof this labile terminator and the demonstration of a one-cyclestop-start DNA synthesis identified the initial steps in the developmentof a novel sequencing strategy. The major challenge for SNP detectionusing BASS, however, is the continued synthesis and identification ofnovel 3′-modified nucleotides that give the desired properties oftermination with removable protecting groups.

[0131] 4. Using Single Base-modified 3′-dNTPs

[0132] Kornher and Livak (1989) described another method byincorporating mobility shifting modified-dNTPs (i.e., the attachment ofa biotin group or a fluorescein group to the base) into a PCR amplifiedDNA sample. The SNP is identified by denaturing gel electrophoresis byobserving a “slower” migrating band, which corresponds to theincorporated modified nucleotide into the DNA fragment.

[0133] 5. Using Single Alpha-thio-dNTPs

[0134] Other methods that can be employed to determine the identity of anucleotide present at a polymorphic site utilize modifiedalpha-thio-dNTPs, which are resistant to exonuclease cleavage (U.S. Pat.No. 4,656,127). An oligonucleotide, of identical sequence to acomplementary target region, immediately flanks the 5′-position of thesingle nucleotide mutation site under investigation. If the polymorphicsite on the DNA contains a nucleotide that is complementary to theparticular exonuclease-resistant nucleotide derivative present, thenthat derivative will be incorporated by a polymerase and extend theoligonucleotide by one base. Such incorporation makes the primerresistant to exonuclease cleavage and thereby permits its detection. Asthe identity of the exonuclease-resistant nucleotide derivative is knownone can determine the specific nucleotide present in the polymorphicsite of the DNA.

[0135] 6. Using Single Labeled 2′,3′-dideoxynucleotides

[0136] Several groups have reported methods for single nucleotideincorporation assays using labeled 2′,3′-dideoxynucleotides tospecifically assay given SNPs of interest, which are detected byautoradiography, calorimetrically, or fluorescently (Lee and Anvret,1991; Livak and Hainer, 1994; Nikiforov et al., 1994; Shumaker et al.,1996). All of these methods rely on PCR to amplify genomic DNA frompatient material and careful design of oligonucleotide sequences totarget specific known mutations in different genes. The separation ofdideoxynucleotide incorporated DNA fragments can be achievedelectrophoretically (Lee and Anvret, 1991; Livak and Hainer, 1994;Nikiforov et al., 1994; Shumaker et al., 1996) or by using high-densityarray chip formats (Shumaker et al., 1996).

[0137] 7. By Direct Detection from Genomic DNA

[0138] One aspect of this invention is to develop a DNA sequencingdevice for analyzing SNPs directly from genomic DNA without cloning orPCR amplification. The present invention circumvents the problemsassociated with the previously described methods for SNP detection,which rely on a prior PCR or cloning step. These steps potentially adderrors in sample handling, introduction of exogenous contamination,significant costs in reagents and labor and seriously hamper theintroduction of high-throughput SNP detection into a clinical or medicalsetting. Because of its simplicity, the PME technology has thecapability of greatly increasing the multiplexing of numerous SNPssimultaneously, which is significantly limited in other previouslydescribed systems. For example, 4-, 8-, 12- and 16-differentfluorophores identified herein can be coupled to appropriateribonucleotides, 2′-deoxynucleotides, 2′,3′-dideoxynucleotides,2′3′-unsaturated-dNTPs and/or other modified nucleotides.

[0139] Moreover, the assay can be multiplexed, and coupled to ahigh-throughput electrophoresis system, and in this configuration, ithas the capability of analyzing 2,000-to-4,000 independent SNPs inapproximately 30-to-60 minutes. Multiplexing is accomplished by varyingthe length of the specific primers by increments of 2-to-3 bases, and byincreasing the number of fluorophores detected in the SNP assay. Twentyto 100, or more specifically 30-to-50 primers, all differing in length,could be assayed in a single nucleotide primer extension assay, whichcould be resolved by electrophoresis and detected by PME in a singlecapillary. Since the longest primer sequence will generally not exceed100 bases in length, fast separation times are expected. It isnoteworthy that SNP specific primers can also be arrayed in ahigh-density chip format, thus eliminating the need for electrophoresis.The scalability of the DNA sequencer is multiplied by 96-capillaries.

[0140] C. Massively Parallel Signature Sequencing (MPSS) Strategy

[0141] Brenner et al. (2000) recently presented data on their massivelyparallel signature sequencing (MPSS) strategy, which is another cyclicprocess involving type II restriction digestion/ligation/hybridizationto sequence over 269,000 signatures of 16-20 bases in length (Brenner etal., 2000). The main disadvantage of MPSS is low efficiency where only25% of the starting DNA templates yield signatures after application ofthe base-calling algorithms.

[0142] d. Ligase Chain Reaction (LCR)

[0143] LCR can also amplify short DNA regions of interest by iterativecycles of denaturation and annealing/ligation steps (Barany, 1991). LCRutilizes four primers, two adjacent ones that specifically hybridize toone strand of target DNA and a complementary set of adjacent primersthat hybridize to the opposite strand. LCR primers must contain a 5′-endphosphate group such that thermostable ligase (Barany, 1991) can jointhe 3′-end hydroxyl group of the upstream primer to the 5′-end phosphategroup of the downstream primer. Successful ligations of adjacent primerscan subsequently act as the LCR template resulting in an exponentialamplification of the target region. LCR is well suited for the detectionof SNPs since a single-nucleotide mismatch at the 3′-end of the upstreamprimer will not ligate and amplify, thus discriminating it from thecorrect base. Any or all of the LCR primers can be labeled withdifferent fluorescent dyes for unambiguous discrimination of specificSNPs. Although LCR is generally not quantitative, linear amplificationsusing one set of adjacent primers, called the Ligase Detection Reaction,can be quantitative. Coupled to PCR, linear ligation assays can also beused as a mutation detection system for the identification of SNPs usingboth wild-type-specific and mutant-specific primers in separatereactions.

[0144] e. Oligonucleotide Ligation Assay (OLA)

[0145] The Oligonucleotide Ligation Assay was first reported to detectSNPs from both cloned and clinical materials using a 5′-end biotin groupattached to the upstream primer and a nonisotopic label attached to thedownstream primer (Landegren et al., 1988). Allele-specifichybridizations and ligations can be separated by immobilization to astreptavidin-coated solid support and directly imaged under appropriateconditions without the need for gel electrophoretic analysis.Subsequently, Nickerson et al. have described an automated PCR/OLAmethod for the diagnosis of several common genetic diseases. FollowingPCR amplification, the upstream 5′-end biotinylated primer anddigoxigenin labeled downstream primer are ligated together underappropriate and specific annealing conditions, captured on streptavidincoated microtiter plates and detected calorimetrically by an alkalinephosphatase assay (Nickerson et al., 1990)

[0146] f. Ligase/Polymerase-Mediated Genetic Bit Analysis

[0147] U.S. Pat. No. 5,952,174 describes a method that also involves twoprimers capable of hybridizing to abutting sequences of a targetmolecule. The hybridized product is formed on a solid support to whichthe target is immobilized. Here the hybridization occurs such that theprimers are separated from one another by a space of a singlenucleotide. Incubating this hybridized product in the presence of apolymerase, a ligase, and a nucleoside triphosphate mixture containingat least one deoxynucleoside triphosphate allows the ligation of anypair of abutting hybridized oligonucleotides. Addition of a ligaseresults in two events required to generate a signal, that is extensionand ligation. This provides a higher specificity and lower “noise” thanmethods using either extension or ligation alone and unlike thepolymerase-based assays, this method enhances the specificity of thepolymerase step by combining it with a second hybridization and aligation step for a signal to be attached to the solid phase.

[0148] XI. Data acquisition and analysis

[0149] The PME system, including switching the lasers and collecting thedata are under computer control in a unified hardware/softwareframework. Cross-platform versatility is achieved, for example, by usingPCI-bus data acquisition and controller cards and LabView™ software fromNational Instruments. The graphically oriented acquisition and analysisenvironment provided by LabView has led to its widespread adoption inlaboratory use. Software programs have been developed to perform severaloperations for the PME sequencing prototypes including: (i) generating atrigger signal for the TTL clock chip to govern the basic 4 sub-cycles(serial pulsing of lasers 1 through 4) of each cycle, (ii) controllingthe blocking of scattered light, (iii) acquisition of thetime-integrated signals from the photodetector, and (iv) controllingvarious operations for automated capillary electrophoresis methods.Scattered light is controlled by use of a liquid crystal tunable filterunder electronic command (e.g., the VariSpec from CRI, Inc.) to providea different edge block for each of the four lasers. When using a PMT oravalanche photodiode, sampling should be taken several times persub-cycle for purposes of time-integration. For the full-scalemultichannel CCD operation, only one read per sub-cycle is necessary.These different modes of operation are easily handled in software. Asmentioned above, a primary goal of this invention is direct detection,which would eliminate the need for PCR amplification. This requires highdirect sensitivity such as can be obtained with a spectroscopic-gradeCCD camera with very low readout noise (e.g., a few accumulated photonsper readout).

[0150] In addition, software programs that perform a number of dataanalysis steps, including spectral matrix correction, baselinecorrection, electrophoretic mobility corrections, base-calling of thesingle nucleotide, quantitation of peak heights for heterozygoteanalyses, allele association by electrophoretic position and order ofdifferent fluorescently labeled gene targeted primers are developed.Excitation by PME produces some level of cross-talk from the non-matchedlaser pulses other than from the best matched laser. As discussedpreviously, laser-dye combinations that minimize non-matched lasercross-talk can be easily identified, so that time correlated excitationof the correct fluorophore can be identified and made with highconfidence. In general, however, it will be necessary to accommodateheterozygous base pairs, particularly for SNP analyses in which morethan one fluorophore is excited at a time. Under non-saturatingconditions, this leads to linear relations between the number N_(i) ofphotons detected due to illumination by laser i and the number n_(j) ofmolecules of dye j,

N _(i)=Σ_(j)α_(1,j) n _(j)

[0151] The matrix α implicitly contains factors including the molarextinction coefficients of the different dyes at frequency i, theirquantum yields, the efficiencies of the laser and the detection system,and attenuation effects. From a practical point of view, the relativemagnitudes of the elements α_(i,j) are calibrated experimentally. Thematching of the dye maxima to the laser colors makes the matrixdiagonally dominant, allowing it to be inverted without numericaldifficulties. The inversion of α removes the residual cross-talk betweenthe dyes. Thus it should be possible to directly obtain the relativenumbers of dye molecules with maximum contrast from the four-colorexperiments. Corrections to this may come from scattered light. While asimple baseline correction is easily accommodated, light fluctuationswill add some noise to the experiments. Several full cycles of the fourlasers will pass during each elution component, allowing reduction ofthe noise by signal averaging. At the same time, the quantification ofthe noise provides a real-time diagnostic for estimating confidencelevels on the signal measurements. Base-calls should in any case proceedwith high confidence since the precise handling of the cross-talk willordinarily yield one dye population that is much higher than the others.In those cases where heterozygotes are present, it is straightforward todistinguish these mixed-populations since they will yield two dyes withhigher (and approximately equal) populations.

[0152] As used herein, the term “timing program” is meant to includeeither software or hardware configured to signal a laser firingsequence. The timing program will also contain information from thelaser firing sequence, which can be correlated with the fluorescenceemission signal.

[0153] As used herein, the term “excitation line” means a laser beam oroutput from another excitation source having a spectral wavelength orits corresponding frequency.

[0154] As used herein, the term “substantially all” means at least 90%.For example, “substantially all of the fluorescence signal” is at least90% of the signal.

[0155] As used herein, the term “substantially corresponds” means thatthe difference between the two is less than 5%. For differences inwavelengths in the visible spectrum, this corresponds to differences of20-33 nm, or more preferably 10-20 nm, or even more preferably 5-10 nm,or most preferably, when the absorption maxima of a dye “substantiallycorresponds” to the excitation wavelength of an excitation line, the twowavelengths are less than 5 nm.

[0156] As used herein, the term “optically matched” means that thewavelength maxima are within one nm of each other.

[0157] The term “substantially colinear” means that the laser beams orexcitation lines diverge from each other at angles of less than 5°.

[0158] The term “substantially coaxial” means that the laser beams orexcitation lines diverge from each other at angles of less than 5°.

[0159] The term “phased shifted,” means that the phase relationshipbetween two alternating quantities of the same frequency is changed. Forexample, consider two trains of repeating pulses such as pulse train (1)laser 1 on followed by laser 1 off for three times as long and pulsetrain (2) laser 2 on followed by laser 2 off for three times as long.One would say that the sequence of equal time periods of laser 1 on,laser 1 off, laser 2 on, laser 2 off corresponds to the sum of pulsetrain (1) and pulse train (2) with the phase of pulse train (2) delayedby a phase shift of 180° or a half cycle. Note that for 180°, delayed oradvanced phase shifts are equivalent.

[0160] As used herein, the term an “on-time window” is defined as thewindow of time corresponding to when the excitation line is incident onthe sample and includes the window of time corresponding to the timeafter the excitation line has ceased firing and before a secondexcitation line is incident on the sample.

[0161] As used herein the specification, “a” or “an” may mean one ormore. As used herein in the claim(s), when used in conjunction with theword “comprising” , the words “a” or “an” may mean one or more than one.As used herein “another” may mean at least a second or more.

[0162] XII. EXAMPLES

[0163] The following example is included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which follow,represent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Optical system

[0164] To test the concept of the PME system, a simple breadboard devicewas built to test the feasibility of discriminating differentfluorescent signals from a mixture of two BODIPY fluorophores (Metzkeret al., 1996). The optical path for combining the pulsed 532 nm and 635nm lines is depicted schematically in FIG. 1 as solid lines. The laserlight emitted from the green 532 rim solid state, diode-pumped,frequency-doubled Nd:YAG laser (Intelite, Minden, N.Y.) and the red 635nm SPMT diode laser module with external potentiometer (Blue SkyResearch, San Jose, Cailf.) were each directed using two commercialgrade aluminum steering mirrors (Edmund Industrial Optics, Barrington,N.J.) to a dual prism assembly. The prisms were coated with a singlelayer HEBBAR antireflection material, which reduced polarization at theprism surfaces by increasing total transmittance. The high dispersionequilateral prisms were constructed from F2, grade “A” fine annealedflint glass and were positioned at a forty-five degree angle relative toone another to allow efficient overlap of the two beams by inversedispersion into a single beam, FIG. 2. The flexibility of this designallows as many as eight excitation lines originating from discrete pointsources (five lasers are shown in FIG. 1 for illustration) to becombined efficiently by the inverse dispersion strategy.

[0165] The combined laser beams were directed into the cuvette assemblybox, which consists of a hollow aluminum lightproof box. The box wasmodified by affixing two “floating” adjustable iris fixtures (EdmundIndustrial Optics) to minimize the amount of stray light entering thebox. A 10 cm cylindrical optically correct glass cuvette (NSG PrecisionCells, Inc, Farmingdale, N.Y.) was installed and mounted using two blackdelrin holders. A 500K multi-alkali PMT detector, which has goodsensitivity in the range of 280 nm to 850 nm, was coupled to the cuvetteassembly box. The fluorescence was detected directly from the cuvetteusing a collection lens in an orthogonal geometry to the propagationdirection of the excitation laser beams.

Example 2 Pulse Generation System

[0166] There are a number of methods to serially pulse multiple lasers,including mechanical chopping and TTL control. One strategy was toserially pulse the 532 nm solid-state laser and the 635 nm diode laserby TTL control using 74174-clock chip, (FIG. 3). The advantages of theclock chip TTL circuit are its simplicity and flexibility as it isdesigned to pulse of up to eight discrete sources. As an alternative, aTTL bucket brigade circuit was constructed because of its simplisticdesign in pulsing 4-lasers using 2 dual J/K flip/flop chips. The TTLClock Chip essentially provides a means for distributing the timingpulses from a master clock on the computer to the appropriate lasers. Aseach clock pulse is received, the chip output sequentially shifts onestep from Q0 to Q1 . . . . and finally to Q7 on the 8th clock pulse.Each laser is turned on in turn for one and only one clock pulse. Thetotal cycle time may be easily varied by several orders of magnitude,from tens of seconds to milliseconds simply by changing the master clockfrequency, and this has been successfully tested. The duration of thelaser pulses are always identical to each other, and the off timebetween the pulses remain in the same exact proportion to each other, sothe overall cycle time may be changed with a single parameter.

Example 3 Results and Discussion from the 2 Color PME Study

[0167] A preliminary experiment was performed to determine thefeasibility of the PME approach to discriminate each fluorophore from amixture of fluorescence dyes. To test the concept of “colorblind”detection, this experiment was performed without the aid of fluorescenceband pass filters, laser line blocking filters, gratings, prisms, or anyother dispersing elements to aid in distinguishing one dye's emissionfrom the other. Moreover, the raw output from the photomultiplier wassent directly to an oscilloscope, without signal averaging or any othertype of processing enhancement. Each laser was alternately pulsed for1.2 msec and was configured with the red laser connected to Q0 and thegreen laser connected to Q3, FIG. 3. Altering the green laser to Q2 gavethe correct firing sequence, which verified the proper configuration ofthe TTL circuit (data not shown). The remaining Q inputs were idle andresulted in dark spacing between laser pulses of 2.4 msec (red-to-green)and of 4.8 msec (green-to-red). The red and green lasers provided 5.5 mWand 4 mW of power, respectively. The difference in power settings waspurposeful to partially offset the higher detector sensitivity of thegreen fluorescence over the red fluorescence. Photographs were taken bya digital camera in real time, and the fluorescent signals were recordedin a downward (negative) direction, as the PMT multiplies electrons.

[0168] The two dyes examined were BODIPY (523/547) and BODIPY (630/650),which have narrow absorption/emission half-bandwidths (Metzker et al.,1996), and therefore are ideal for the color-blind PME detection scheme.The dyes were analyzed at a concentration of approximately 10⁻⁵ M inethanol. This moderately high concentration was chosen to assure thatthe signals were derived entirely from the dye solutions, and not fromother sources, such as stray light, thus providing an accurate measureof the contrast ratio. The emission from each dye fell in toto onto thered-sensitive photomultiplier, which is a key feature of the color-blindmethodology.

[0169] In FIG. 4A, the oscilloscope trace was obtained from an equalmixture of BODIPY dyes in the cuvette. The two channels from theoscilloscope photographs were set to record the trigger signal, whichturns on the red laser (upper trace) and the fluorescence signal fromthe PMT detector (lower trace). The green laser pulse was subjected to apartial modulation, which gives the fluorescence output a “two-finger”appearance, making it easily distinguishable from the smooth red laserfluorescence. The small variation observed in the red laserpulse-to-pulse fluorescence intensity was due to 120 Hz leakage from aninadequately rectified AC power supply, and can be corrected byinsertion of a capacitor π filter in the power supply in theexperimental section. As shown, the timing of the sequential firing ofthe red laser and then the green laser resulted in significantfluorescence signals when both BODIPY dyes were present in solution.

[0170]FIG. 4B shows the total fluorescence signal from the serialpulsing of the green and red lasers with only the BODIPY 630/650 (reddye) present in the cuvette. Whereas the large fluorescence signal istime correlated with the firing of the red laser, the green laser onlyimparts a small “cross-talk” signal to the red dye. This cross-talk wasmeasured to be approximately 4% of the red laser signal, which isattributed to the highly efficient coupling of a laser precisely matchedto the red dye absorption peak and the narrower absorption spectralproperties of BODIPY dyes. Both aspects give the desired low excitationefficiency of the off-resonant green laser. This result clearlyillustrates good contrast between laser excitations for the same dyeusing the PME approach. Since the ratio of red to green excitationefficiency can be determined, a cross-talk matrix can be computed andmathematically applied to yield a much higher contrast ratio.

[0171]FIG. 4C shows the total fluorescence signal from the serialpulsing of the red and green lasers with only the BODIPY 523/547 (greendye) present in the cuvette. The only fluorescence signal observed istime correlated with the firing of the green laser, which gives the“two-finger” signature. Unlike that of the BODIPY 630/650, thecross-talk signal observed from the red laser is negligible on thisscale, and further signal amplification revealed it to be considerablyless than 1% of that from the green laser. This observation is expectedbecause longer wavelength excitation sources should not impose photonabsorption on shorter wavelength dyes (i.e., the red laser does notexcite the green dye). This feature illustrates an important and keyadvantage of reduced cross-talk of fluorophores using the PME strategy.Consider a four dye system, which is made-up of blue, green, yellow, andred dyes. The blue dye should not exhibit cross-talk from the sequentialfiring of the green, yellow, and red lasers. The green dye, on the otherhand, will exhibit cross-talk from the only blue laser, but not theyellow or red lasers. The yellow dye will exhibit cross-talk from theblue and green lasers, but not the red laser and so forth. In otherwords, the observed cross-talk on the “blue” portion of the spectrumrelative to the absorption/excitation maxima of a given dye is negated,which is significantly different from emission cross-talk of blue green,yellow, and red dyes excited using a single excitation source.

[0172] An excellent contrast ratio for the discrimination of twodifferent BODIPY dyes using the PME technology by detecting all of thefluorescence emission in a true color-blind fashion is demonstrated. Theexperiment was designed using no spectral filtering elements of anykind, and the signal was taken directly from the oscilloscope in realtime without signal averaging or other processing. These data show thatthe choice of experimental conditions provided significant fluorescencesignal for which scattered laser light signals are negligible.Therefore, the 25:1 contrast ratio observed for this unaveraged rawsignal obtained with laser pulses on the msec timescale provides agenuine comparison of the time correlated fluorescence detectiontechnique.

Prophetic Example 4 Development of a 1-capillary 4-color PME Prototype

[0173] a. Develop 4-color: Identification of the Optimal 4 Laser-dyeCombination

[0174] Six different solid-state lasers and/or laser diode modules havebeen identified with excitation wavelengths that match the absorptionmaxima of a number of commercially available fluorophores, most of whichhave been used for DNA sequencing. Other lasers and fluorophores canalso be identified by comparing and matching the excitation maxima ofthe fluorophore with the emission wavelength of the laser. Candidatedyes should show good quantum yields and have narrow absorption spectra.The non-limiting list of dyes listed herein below initially meet theserequirements, although other commercially available fluorophores andlasers may be tested as well. For experimentation purposes, each dye iscoupled to the universal sequencing primer (5′-TTGTAAAACGACGGCCAGT)(Metzker et al., 1996) as representative of termination products for theSNP assay. TABLE 1 Laser Fluorophore Blue 399 nm solid state,7-dimethylaminocoumarin (409/473), indium gallium nitride laser cascadeblue (396/410), and 7- hydroxycoumarin (386/448). Blue 473 nm or 488 nm5-carboxyfluorescein (494/518), 1,3,5,7- solid state, diode-pumped,tetramethyl-BODIPY (495/503), Oregon frequency-doubled green 488(496/524), and the 5,7-dimethyl- Nd:YAG laser BODIPY (503/512).* Green532 nm solid state, BODIPY (523/547) (536/554 when coupled diode-pumped,frequency- to a primer) doubled Nd:YAG laser Yellow 594 nm He-Ne BODIPY589/617 and BODIPY 581/591 (592/603 when coupled to a primer) Red 635 nmSPMT diode BODIPY 630/650 (643/651 when coupled laser module withexternal to a primer) potentiometer Red 670 nm SPMT diode The BODIPY(650/665) dye (661/667 when laser module with external coupled to aprimer) and cyanine 5 dye potentiometer

[0175] Although some dyes listed in Table 1 have a listed absorptionmaxima on the blue side of the excitation source, these dyes will beconsidered for use since the attachment of dyes to DNA usually resultsin a red shift in the absorption/emission spectra. Theabsorption/emission values for the dyes given in parenthesis correspondto the absorption and emission wavelengths when coupled to a universalsequencing primer.

[0176] A systematic evaluation of each laser-dye combination will becompared for good excitation characteristics and between laser-dye pairsto identify an optimal set of 4 laser-dyes for DNA sequencingapplications. Each laser will be set-up, similar to the green and redlaser experiment and pulsed using the TTL clock chip for excitation andcross-talk experiments as described in Example 3. It is anticipated thatnumerous new solid-state lasers and laser diodes and new fluorescentdyes will continue to be developed and commercialized with uniqueemission wavelengths ranging below 400 nm to beyond 1100 nm that can beused in the current invention. Due to the modular configuration of thePME system, the testing of additional laser-dye pairs isstraightforward.

[0177] b. Construction of the 1-capillary Breadboard Prototype

[0178] A 1-capillary electrophoresis unit will be set-up on a breadboardplatform, and the electrophoresis will be driven initially using a 30kVpower supply. A Plexiglas box equipped with a safety interlock will beconstructed to enclose the samples and the running buffers. Initially,electrophoresis will be performed under ambient conditions due to thenature of the short primer extension products; however, a temperaturecontrolled heating jacket to improve electrophoretic resolution will beconstructed if necessary. The separation format will use fused silicacapillaries (150 μM OD, 50 μM ID, and 50 cm in length), POP-6 solutionas the separation matrix, and TBE as the running buffer. A 1-capillarysheath flow cuvette will be constructed using the rectangular, tapereddesign described by Zhang et al. (1999) and sheath flow will be drivenby syringe pump at a flow rate of approximately 0.3 mL per hour.

[0179] C. Sensitivity Experiments for Direct Detection

[0180] Although limited sensitivity information can be obtained from the2-color PME system (FIG. 1), more data will be obtained from conductingsensitivity experiments directly using the 1-capillary instrument.Subsequent to the identification of the 4 laser-dye set, but overlappingwith the construction of the 1-capillary instrument, the PME lasersystem will be coupled to the electrophoresis device. For limit ofdetection assays, both the PMT and the silicon APD (current and countingmodes) will be investigated over a wide range of fluorophore-labeleduniversal primer concentrations. Sensitivity assays will be conductedusing free zone and POP-6-based capillary electrophoresis. A uniquefeature of the PME system is that limit of detection experiments will beperformed for the 4 laser-dye sets identified previously. It should benoted that limit of detection experiments are only informative regardingsensitivity when the test dye is optimally excited and producing maximumfluorescence signal. Sensitivity experiments are not possible orpractical using all four fluorophores with the standard spectrallyresolved DNA sequencing systems because the longer wavelength dyes areinefficiently excited. Therefore, the limit of detection experimentstypically published in the literature are performed using the dye mostclosely matched to the laser source (out of the set of four), which isusually fluorescein and the argon ion laser (Swerdlow et al., 1990;Drossman et al., 1990; Swerdlow et al., 1990; Zhang et al., 1999). Here,limit of detection experiments will be performed using all four PMEfluorophores because the lasers are closely matched for optimalexcitation and therefore will produce a more robust picture forsensitivity with respect to the entire sequencing chemistry.

[0181] d. Development of Rapid Sample Preparation Methods for DirectFluorescent Assays from Genomic DNA

[0182] The PME instrument should be able to detect as few as 10⁴-to-10⁵fluorescent molecules. Given that 1 mL of whole blood containsapproximately 10⁶ white blood cells, direct detection (without the needfor amplification of the sample) of multiple SNPs is possible.Initially, SNP assays will be performed using standard PCR techniquesand diluted appropriately to simulate direct genomic DNA levels. Thisapproach will allow the performance of limit of detection experimentswithout dependence or delay for the development of optimized samplepreparation methods and direct genomic SNP assays.

[0183] Sample preparation methods will be developed from whole blood tobe fast, simple, and amenable to direct single nucleotide primerextension assays. Typically, most methods involve the fractionation ofwhole blood into serum, red blood cells, and white blood cells, of whichthe latter is used for analysis. Sample preparation experiments can relyon commercially available kits and published protocols for evaluationand optimization.

[0184] As discussed hereinabove, sequencing assays are typicallyperformed in μL quantities, but loaded onto commercial capillaryelectrophoresis instruments in nL quantities. To minimize wasting directassay samples, reaction volume assays will be optimized in a targetvolume of 1 μL. Since electrokinetic injection will be implemented asthe injection method for each prototype, injection biases will mostlikely occur depended on sample purity (Huang et al., 1988). Therefore,several solid-phase and affinity-based purification schemes will beinvestigated for producing highly purified fluorescently labeled SNPassays, which are devoid of contaminants, such as unincorporatedfluorescent terminators, salts, and other electro-competingmacromolecules.

[0185] In the event that the sensitivity limit of the PME technology isseveral orders of magnitude higher than anticipated (i.e., 10⁶-to-10⁷fluorescent molecules), direct detection from whole blood can still beachieved. This can be done by increasing the amount of blood analyzedfrom 1-to-10 mL, and/or performing a linear amplification of the primerextension assay by temperature cycling, typically used in Sangersequencing reactions.

Prophetic Example 5 Construction of a Portable 8-capillary PME DNASequencer

[0186] a. Construction of 8-APD Detector for a Portable System

[0187] A PME DNA sequencer that is portable will be useful for anyapplications where there are space limitations or where it is importantto be able to move the sequencer. The technology to modify the PME DNAsequencer such that it is portable is currently available. For theportable DNA sequencer, the optical system developed for the breadboardwill be adapted to become more compact and robust. The highly efficientdual prism combiner will be initially adapted to the portable sequencer.Constructed with microbench components, the optics train will beincorporated into a 4-rail structure, which was developed for the veryrigid needs of laser cavity mirror supports. The commercially availableminiature 4-rail system will also be used to support the sheath flowcuvette and collection optics. As previously discussed, commercial diodelaser modules are remarkably compact, typically 1″or 2″long, and aregenerally available with optical fiber coupled outputs. Fibersplitter/combiners have been developed for laser based communications,and contingent on this rapidly emerging technology, it may be feasibleto combine the beams and deliver the 4-laser sources to the sheath flowcuvette in a single optical fiber. For this design, only a conventionalachromatic lens will be needed to project the collimated alternatingmulticolor beam through the sheath flow cuvette.

[0188] A wide field f/1 (NA ˜0.5) lens will be used to collect thefluorescent light from the capillary plumes and project it onto 8 APDs.A microscope objective is often used for this purpose, but may suffervignetting and consequent loss of fluorescence signal from the outermostcapillary plumes. The lens mount will be equipped with opposingadjustment screws that lock the lens into place after optimization. Thelight path will be shielded with baffles to reduce scattered laser lightreaching the detectors. An optional liquid crystal device will be usedas an edge filter to block scattered laser light, as needed; this devicehas a rejection ratio of about 4 orders of magnitude. Unlike thefamiliar rotating filter wheel, the liquid crystal device has nomechanical moving parts. The liquid crystal filter has a response timeof several milliseconds, so that it will be cycled under computercontrol along with the 4 lasers, blocking scattered light from each onein turn, while passing essentially all of the fluorescent light to thecolor blind APD detectors.

[0189] Although it is preferable to imaged fluorescent light directlyonto the APDs, the collection lens, which typically magnifies the image20× may not be enough to resolve 8 images for detection. One solution isto use a small mirror or prism affixed to each APD housing, which candeflect the beam at right angles. This design can be mounted in astaggered array and thereby reduce this congestion. Individual gradientrefractive index (GRIN) lenses and optical fiber couplings to each APDwill also be considered in the portable configuration. However, theinsertion loss for a properly coated beam steering prism is about 2%,and it is most unlikely that the fiber coupling will perform as well.This fiber coupling problem is much more significant for the projectedfluorescent image, which behaves as an extended source, than it is for alaser beam. Finally, the same rigid 4-rail structure mentioned abovewill be used to support the detection system.

[0190] The laser and TTL circuit power supplies typically have afootprint of a few square inches, and the power supplies for the APDsand the 10 kV electrophoresis modules are slightly larger, but stillonly a few inches long. These various electronic components will beeasily positioned beneath and around the capillaries, which will travelaround the perimeter of the system, thus avoiding sharp bends.

[0191] b. PME of Residual Signal

[0192] Residual fluorescence may be detected immediately after veryshort excitation laser pulses have irradiated the sample. With such anapproach, the lasers will be off during the period that the fluorescentsignal is collected. This may be particularly important for thedevelopment of sequencing on a chip, because the chip almost inevitablywill generate large amounts of scattered light.

[0193] Specifically the intent is to sequentially fire picosecond laserpulses at a fluorescently labeled DNA sample and then “look” for afluorescent response on the nanosecond time-scale, immediately after thelaser pulse ends. This novel approach is a logical extension to thecentral principle of operation intrinsic to the core PME technology.This innovative experimental strategy is referred to as “Looking In TheDark” or “PME-LITD”.

[0194] The primary advantage of the Looking In The Dark strategy is thecomplete elimination of scattered light, which at low levels offluorescence is likely to be a main source of noise in the PMEinstrument. This technique, if successful, could have enormousimplications for improving the signal-to-noise ratio and potentiallyimprove the overall sensitivity of the instrument.

[0195] The following example details a simple sequence of events toillustrate the PME-LITD concept:

[0196] 1. The first laser in sequence is pulsed for 50 pico-seconds.

[0197] 2. A 500 pico-second time delay is applied after the laser hasbeen switched off Note that during the delay period no fluorescence issampled by the detector.

[0198] 3. A fast photon counter is used to look for any fluorescentresponse from the labeled DNA during the ensuing 50 nano-second gatedwindow.

[0199] 4. Steps 1 through 3 are repeated in sequence for each laser inthe subcycle.

[0200] 5. The picosecond pulsed excitation and nano-second gateddetection windows cycle continuously.

[0201] For a four color system, the above steps would generate asubcycle time that is 202.2 nanoseconds. This implies that over an eightsecond time window, (approximate time for a labeled DNA band to passthrough an ABI 3700 cuvette), around 40 million complete subcycles wouldbe completed. The data collected will then be appropriately averaged andfurther processed to yield high quality analyzed data.

[0202] To conduct these types of experiments, lasers that are capable ofgenerating very short pulses of sub-nanosecond duration will berequired. Pico-second laser sources are commercially available from anumber of companies including Coherent Laser Group, Newport, andPicoQuant. For example, Coherent has a diode pumped mode-locked laserwith fundamental wavelengths at either 1047 nm, 1053 nm or 1064 nm,which generates pulses as short as 2 ps. The second harmonics can easilybe generated from picosecond sources, hence the following wavelengthswould be available: 532 nm, 526.5 nm, and 523.5 nm. Newport alsomanufactures a “NanoLaser” that generates sub-nanosecond green, (532 nm)light, at an average power of more than 6 mW.

[0203] In addition, an instrument that is capable of counting photons ona sub-nanosecond time scale will also be needed; such devices areavailable from a variety of manufacturers. For example, “FAST ComTec”produces a single photon counting instrument, which has resolution onthe time-scale of 500 pico-seconds. Becker & Hickl GmbH alsomanufactures a four channel correlated single photon counting devicethat has resolution down to 813 femto-seconds. Furthermore, it should benoted that when coupled into an appropriately configured electroniccircuit the recovery time of a silicon avalanche photo-diode detector,(following illumination by scattered light from a excitation laserpulse), is of the order of 500 pico-seconds. This rapid recovery timewill permit the effective observation of fluorescence from a dye with afluorescent lifetime of several nanoseconds in the complete absence ofany laser excitation, i.e. “in the dark”.

[0204] Four wavelengths can be generated from a single laser, as opposedto using four synchronized and mode-locked lasers. This can be doneusing Stimulated Raman Shifting, (SRS).

[0205] An experiment to test the PME-LITD strategy comprises a simpletwo-color system. Specifically, a mode-locked Nd:YAG laser generating 50pico-second pulses will be coupled to a Raman cell filled with molecularnitrogen. The superimposed multi-wavelength output from the Raman cellwill be then dispersed and ultimately recombined using a four-prismassembly. In the middle of the four-prism assembly, (i.e. where thevarious excitation lines are separated and traveling approximately inparallel), a pair of electro-optic modulators will be used to chop thecolored pulses—selecting alternate pulses from each beam. The recombinedbeams will then be directed into a cuvette assembly—similar to theprototype described in FIG. 1. Finally, the time-resolved fluorescencewill be detected using a fast photon counter that looks for photons in awindow that spans the range from 0.5 ns-50.5 ns after the cessation ofeach laser pulse.

[0206] C. Construction of a 96-capillary PME Suitcase DNA Sequencer

[0207] A portable 96-capillary PME DNA sequencer is envisioned as anaspect of the current invention. In one embodiment, the four-laserillumination system described in the 8-capillary sheath flow cuvettesystem will be used for the 96 capillary system. All four alternatingexcitation lines will be coaxial and well collimated to facilitate theillumination of the 96 fluorescent plumes. Although the compactmulti-laser source will remain unchanged, it will not be practical toscale the detection system from 8 APDs to 96 discrete detectors. A CCDcamera, however, will be more suited to perform this operation. A fastlens such as f/1, with good imaging quality will be installed forefficient light collection. A second lens will be used to re-image thelight onto the CCD. The computer-controlled liquid crystal filter may beinterposed between the two lenses to block scattered laser light, ifneeded. Baffles will be used to minimize stray light, but there are norestricting apertures that reduce the wide cone angle of collection, ordispersing elements that further attenuate the signal.

[0208] Essentially all of the fluorescent light from one sheath flowplume will fall on one particular group of pixels, and these pixels arebinned together so they are read out as a single unit, which will reducereadout noise. Binning of all of the fluorescence from a capillary intoeffectively one giant pixel provides a single robust signal from eachcapillary plume even when the amount of fluorescing dye is quite small.

[0209] The CCD camera is quite compact, and even with addingthermoelectric cooling to reduce background noise, fitting the CCDdetector into a compact device will not be problematic. A portablecomputer will read out the CCD contents at the end of each laser pulse.For a standard video rate of 30 Hz, the entire cycle frequency of 4lasers will be 7.5 Hz (5 Hz with the incorporation of a liquid crystallaser blocker), and this will allow for the data from dozens of readoutcycles to be signal averaged per one elution event. Following theconstruction of the CCD suitcase system, detailed limit of detectionexperiments will be performed to compare it to the performance of the8-capillary APD suitcase prototype.

[0210] All of the methods disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

References

[0211] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0212] Anazawa, T, S Takahashi, and H Kambara (1996) A capillary arraygel electrophoresis system using multiple laser focusing for DNAsequencing. Anal. Chem. 68: 2699-2704.

[0213] Barany, F (1991) Genetic disease detection and DNA amplificationusing cloned thermostable ligase. Proc. Natl. Acad. Sci. USA 88:189-193.

[0214] Brenner S, M Johnson, J Bridgham, G Golda, D H Lloyd, D Johnson,S Luo, S McCurdy, M Foy, M Ewan, R Roth, D George, S Eletr, G Albrecht,E Vermaas, S R Williams, K Moon, T Burcham, M Pallas, R B DuBridge, JKirchner, K Fearon, J Mao, and K Corcoran. (2000) Gene expressionanalysis by massively parallel signature sequencing (MPSS) on microbeadarrays. Nat Biotechnol 18:630-634.

[0215] Cohen A S, D R Najarian, and B L Karger (1990) Separation andanalysis of DNA sequence reaction products by capillary gelelectrophoresis. J Chromatogr. 516:49-60.

[0216] Crabtree H J, S J Bay, D F Lewis, J Zhang, L D Coulson, G AFitzpatrick, S L Delinger, D J Harrison, and N J Dovichi (2000)Construction and evaluation of a capillary array DNA sequencer based ona micromachined sheath-flow cuvette. Electrophoresis 21:1329-1335.

[0217] Deeb S S, L Fajas, M Nemoto, J Pihlajamaki, L Mykkanen, JKuusisto, M Laakso, W Fujimoto, and J Auwerx (1998) A Pro 12Alasubstitution in PPARγ2 associated with decreased receptor activity,lower body mass index and improved insulin sensitivity. Nature Genet.20:284-287.

[0218] Drossman H, J A Luckey, A J Kostichka, J D'Cunha, and L M Smith.(1990) High-speed separations of DNA sequencing reactions by capillaryelectrophoresis. Anal. Chem. 62:900.

[0219] Effenhauser, et al Anal. Chem., 66:2949-2953, 1994.

[0220] Effenhauser, et al. Anal. Chem., 65:2637-2642, 1993.

[0221] Harrison et al., Science, 261:895-897, 1993.

[0222] Huang X, M J Gordon, and R N Zare (1988) Bias in quantitativecapillary zone electrophoresis caused by electrokinetic sampleinjection. Anal. Chem. 60:375-377.

[0223] Huang X C, M A Quesada, and R A Mathies. (1992) Capillary arrayelectrophoresis using laser-excited confocal fluorescence detection: anapproach to high-speed, high-throughput DNA sequencing. Anal Chem.64:967-972.

[0224] Huang X C, M A Quesada, and R A Mathies. (1992) DNA sequencingusing capillary array electrophoresis. Anal Chem. 64:2149-2154.

[0225] Ju J, C Ruan, C W Fuller, A N Glazer, and R A Mathies (1995)Fluorescence energy transfer dye-labeled primers for DNA sequencing andanalysis. Proc. Natl. Acad. Sci. USA. 92:4347-4351.

[0226] Kambara H and Takahashi S. (1993) Multiple-sheathflow capillaryarray DNA analyser. Nature 361: 565-566.

[0227] Karger A E, J M Harris, and R F Gesteland. (1991) Multiwavelengthfluorescence detection for DNA sequencing using capillaryelectrophoresis. Nucleic Acids Res. 19:4955-4962.

[0228] Kheterpal I, J R Scherer, S M Clark, A Radhakrishnan, J Ju, C LGinther, G F Sensabaugh, and R A Mathies (1996) DNA sequencing using afour-color confocal fluorescence capillary array scanner.Electrophoresis 17:1852-1859.

[0229] Kornher, J S and K J Livak (1989) Mutation detection usingnucleotide analogs that alter electrophoretic mobility. Nucleic AcidsRes. 17:7779-7784.

[0230] Kuppuswamy, M N, J W Hoffmann, C K Kasper, S G Spitzer, S L Groceand S P Bajaj (1991) Single nucleotide primer extension to detectgenetic diseases: Experimental application to hemophilia B (factor IX)and cystic fibrosis genes. Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147.

[0231] Kurg A, Tonisson N, Georgiou I, Shumaker J, Tollett J, MetspaluA., “Arrayed primer extension: solid-phase four-color DNA resequencingand mutation detection technology.” Genet Test 2000;4(1):1-7

[0232] Landegren U, R Kaiser, J Sanders, and L Hood. (1988) Aligase-mediated gene detection technique. Science 241:1077-1080.

[0233] Lee J S, and M Anvret. (1991) Identification of the most commonmutation within the porphobilinogen deaminase gene in Swedish patientswith acute intermittent porphyria. Proc Natl Acad Sci USA.88:10912-10915.

[0234] Lieberwirth U, J Arden-Jacob, K H Drexhage, D P Herten, R Muller,M Neumann, A Schulz, S Siebert, G Sagner, S Klingel, M Sauer, and JWolfium. (1998) Multiplex dye DNA sequencing in capillary gelelectrophoresis by diode laser-based time-resolved fluorescencedetection. Anal Chem. 70:4771-4779.

[0235] Livak K J and J W Hainer. (1994) A microtiter plate assay fordetermining apolipoprotein E genotype and discovery of a rare allele.Hum Mutat. 3:379-385.

[0236] Lu X and E S Yeung. (1995) Optimization of excitation anddetection geometry for multiplexed capillary array electrophoresis ofDNA fragments. Appl. Spectrosc. 49: 605-609.

[0237] Luckey J A, H Drossman, A J Kostichka, D A Mead, J D'Cunha, T BNorris, and L M Smith. (1990) High speed DNA sequencing by capillaryelectrophoresis. Nucleic AcidsRes. 18:4417-4421.

[0238] Luryi, S. CRISP Abstract: Grant Number 5R01HG01487-05. 4 Colorautomated DNA sequencing machine with asynchronous network operation.

[0239] Madabhushi R S (1998) Separation of 4-color DNA sequencingextension products in noncovalently coated capillaries using lowviscosity polymer solutions. Electrophoresis 19:224-230.

[0240] Manz, et al., J. Chromatogr., 593:253-258, 1992.

[0241] Meaburn, J (1976) “Detection and Spectrometry of Faint Light”, D.Reidel, Dordrecht, Holland.

[0242] Metzker M L, J Lu, and R A Gibbs (1996) Electrophoreticallyuniform fluorescent dyes for automated DNA sequencing. Science271:1420-1422.

[0243] Metzker M L, R Raghavachari, S Richards, S E Jacutin, ACivitello, K Burgess, and R A Gibbs. (1994) Termination of DNA synthesisby novel 3′-modified-deoxyribonucleoside 5′-triphosphates. Nucleic AcidsRes. 22:4259-4267.

[0244] Nickerson D A, R Kaiser, S Lappin, J Stewart, L Hood, and ULandegren. (1990) Automated DNA diagnostics using an ELISA-basedoligonucleotide ligation assay. Proc Natl Acad Sci USA. 87:8923-8927.

[0245] Nikiforov T T, R B Rendle, P Goelet, Y H Rogers, M L Kotewicz, SAnderson, G L Trainor, and M R Knapp. (1994) Genetic Bit Analysis: asolid phase method for typing single nucleotide polymorphisms. NucleicAcids Res. 22:4167-4175.

[0246] Nyren P, B Pettersson, and M Uhlen (1993) Solid phase DNAminisequencing by an enzymatic luminometric inorganic pyrophosphatedetection assay. Anal. Biochem. 208:171-175.

[0247] Prezant T R, and N Fischel-Ghodsian. (1992)Trapped-oligonucleotide nucleotide incorporation (TONI) assay, a simplemethod for screening point mutations. Hum Mutat. 1:159-164.

[0248] Prober J M, G L Trainor, R J Dam, F W Hobbs, C W Robertson, R JZagursky, A J Cocuzza, M A Jensen, and K Baumeister. (1987) A system forrapid DNA sequencing with fluorescent chain-terminatingdideoxynucleotides. Science 238:336-341.

[0249] Quesada M A and S Zhang (1996) Multiple capillary DNA sequencerthat uses fiber-optic illumination and detection. Electrophoresis17:1841-1851.

[0250] Quesada M A, H S Dhadwal, D Fisk, and F W Studier (1998)Multi-capillary optical waveguides for DNA sequencing. Electrophoresis19:1415-1427.

[0251] Ronaghi M, M Uhlen, and P Nyren. (1998) A sequencing method basedon real-time pyrophosphate. Science 281:363-365.

[0252] Rosenblum B B, L G Lee, S L Spurgeon, S H Khan, S M Menchen, C RHeiner, and S M Chen. (1997) New dye-labeled terminators for improvedDNA sequencing patterns. Nucleic Acids Res. 25:4500-4504.

[0253] Sambrook et al., “Molecular Cloning,” A Laboratory Manual, 2dEd., Cold Spring Harbor Laboratory Press, New York, 13.7-13.9:1989.

[0254] Shibata K, M Itoh, K Aizawa, S Nagaoka, N Sasaki, P Caminci, HKonno, J Akiyama, K Nishi, T Kitsunai, H Tashiro, M Itoh, N Sumi, YIshii, S Nakamura, M Hazama, T Nishine, A Harada, R Yamamoto, HMatsumoto, S Sakaguchi, T Ikegami, K Kashiwagi, S Fujiwake, K Inoue, andY Togawa (2000) RIKEN integrated sequence analysis (RISA)system-384-format sequencing pipeline with 384 multicapillary sequencer.Genome Res. 10:1757-1771.

[0255] Shumaker J M, A Metspalu, and C T Caskey. (1996) Mutationdetection by solid phase primer extension. Hum Mutat. 7:346-54.

[0256] Smith L M, J Z Sanders, R J Kaiser, P Hughes, C Dodd, C RConnell, C Heiner, S B Kent, and L E Hood (1986) Fluorescence detectionin automated DNA sequence analysis. Nature 321:674-679.

[0257] Sokolov, B P (1990) Primer extension technique for the detectionof single nucleotide in genomic DNA. Nucleic Acids Res. 18:3671.

[0258] Sweedler J V, J B Shear, H A Fishman, R N Zare, and R H Scheller(1991) Fluorescence detection in capillary zone electrophoresis using acharge-coupled device with time-delayed integration. Anal. Chem.63:496-502.

[0259] Swerdlow H and R Gesteland (1990) Capillary gel electrophoresisfor rapid, high resolution DNA sequencing. Nucleic Acids Res. 18:1415-1419.

[0260] Swerdlow H, J Z Zhang, D Y Chen, H R Harke, R Grey, S L Wu, N JDovichi, and C Fuller. (1991) Three DNA sequencing methods usingcapillary gel electrophoresis and laser-induced fluorescence. Anal Chem.63:2835-2841.

[0261] Swerdlow H, S L Wu, H Harke, and N J Dovichi. (1990) Capillarygel electrophoresis for DNA sequencing. Laser-induced fluorescencedetection with the sheath flow. J Chromatogr. 516:61-67.

[0262] Syvanen, A C, K Aalto-Setala, L Haxiu, K Kontula, and HSoderlund. (1990) A primer-guided nucleotide incorporation assay in thegenotyping of Apolipoprotein E. Genomics 8:684-692.

[0263] Takahashi, S, M Katsuhiko, T Anazawa, and H Kambara. (1994)Multiple sheath-flow gel capillary-array electrophoresis for multicolorfluorescent DNA detection. Anal. Chem. 66:1021-1026.

[0264] Taylor J A and E S Yeung. (1993) Multiplexed fluorescencedetector for capillary electrophoresis using axial optical fiberillumination. Anal. Chem. 65:956-960.

[0265] Tsuda et al., Anal. Chem., 62:2149-2152, 1990.

[0266] U.S. Pat. No. 4,582,788

[0267] U.S. Pat. No. 4,656,127

[0268] U.S. Pat. No. 4,683,194

[0269] U.S. Pat. No. 4,683,202

[0270] U.S. Pat. No. 5,296,375

[0271] U.S. Pat. No. 5,304,487

[0272] U.S. Pat. No. 5,784,157

[0273] U.S. Pat. No. 5,846,710

[0274] U.S. Pat. No. 5,856,174

[0275] U.S. Pat. No. 5,888,819

[0276] U.S. Pat. No. 5,904,824

[0277] U.S. Pat. No. 5,991,082

[0278] U.S. Pat. No. 6,004,744

[0279] U.S. Pat. No. 6,013,431

[0280] U.S. Pat. No. 6,038,023

[0281] U.S. Pat. No. 6,139,800

[0282] U.S. Pat. No. 6,153,379

[0283] U.S. Pat. No. 6,215,598

[0284] U.S. Pat. No. 6,226,126

[0285] Ueno K and E S Yeung (1994) Simultaneous monitoring of DNAfragments separated by electrophoresis in a multiplexed array of 100capillaries. Anal. Chem. 66: 1424-1431.

[0286] Wei J, and G P Hemmings G P (2000) The NOTCH4 locus is associatedwith susceptibility to schizophrenia. Nature Genet. 25:376-377.

[0287] Woolley and Mathies, Proc Natl Acad Sci USA, 91:11348-52 1994.

[0288] Yeo G S, I S Farooqi, S Aminian, D J Halsall, R G Stanhope, and SO'Rahilly (1998) A frameshift mutation in MC4R associated withdominantly inherited human obesity. Nature Genet. 20:111-112.

[0289] Zagursky R J and R M McCormick. (1990) DNA sequencing separationsin capillary gels on a modified commercial DNA sequencing instrument.Biotechniques 9: 74-79.

[0290] Zhang J, K O Voss, D F Shaw, K P Roos, D F Lewis, J Yan, R Jiang,H Ren, J Y Hou, Y Fang, X Puyang, H Ahmadzadeh, and N J Dovichi. (1999)A multiple-capillary electrophoresis system for small-scale DNAsequencing and analysis. Nucleic Acids Res. 27: e36.

What is claimed is:
 1. A pulse-multiline excitation apparatus foranalyzing a sample containing one or more fluorescent species,comprising: one or more lasers configured to emit two or more excitationlines, each excitation line having a different wavelength; a timingcircuit coupled to the one or more lasers and configured to generate thetwo or more excitation lines sequentially according to a timing programto produce time-correlated fluorescence emission signals from thesample; a non-dispersive detector positioned to collect thetime-correlated fluorescence emission signals emanating from the sample;and an analyzer coupled to the detector and configured to associate thetime-correlated fluorescence emission signals with the timing program toidentify constituents of the sample.
 2. The apparatus of claim 1,wherein the detector and the analyzer are integral.
 3. The apparatus ofclaim 1, wherein the two or more excitation lines intersect at thesample.
 4. The apparatus of claim 1, wherein the two or more excitationlines are configured so that the two or more excitation lines do notintersect in the sample.
 5. The apparatus of claim 1, wherein the two ormore excitation lines are configured so that the two or more excitationlines are coaxial.
 6. The apparatus of claim 1, further comprising anassembly of one or more prisms in operative relation with the one ormore lasers and configured to render radiation of the two or moreexcitation lines substantially colinear.
 7. The apparatus of claim 1,further comprising at least four excitation lines having four excitationwavelengths.
 8. The apparatus of claim 7, further comprising at leasteight excitation lines having eight excitation wavelengths.
 9. Theapparatus of claim 8, further comprising at least sixteen excitationlines having sixteen excitation wavelengths.
 10. The apparatus of claim1, wherein said sample is comprised in at least one capillary.
 11. Theapparatus of claim 1, wherein said sample is comprised in at least 4capillaries.
 12. The apparatus of claim 11, wherein said sample iscomprised in at least 8 capillaries.
 13. The apparatus of claim 12,wherein said sample is comprised in at least 16 capillaries.
 14. Theapparatus of claim 13, wherein said sample is comprised in at least 48capillaries.
 15. The apparatus of claim 14, wherein said sample iscomprised in at least 96 capillaries.
 16. The apparatus of claim 15,wherein said sample is comprised in at least 384 capillaries.
 17. Theapparatus of claim 1, further comprising a sheath flow cuvette.
 18. Theapparatus of claim 1, wherein the timing program comprises a delaybetween the firing of each laser of between about 10 femtosecond andabout 5 seconds.
 19. The apparatus of claim 18, wherein the timingprogram comprises a delay between the firing of each laser of betweenabout 1 millisecond and about 100 milliseconds.
 20. The apparatus ofclaim 18, wherein the timing program comprises a delay between thefiring of each laser of between about 50 ps and about 500 ps.
 21. Theapparatus of claim 1, wherein at least one or more of the excitationlines is pulsed.
 22. The apparatus of claim 21, wherein said pulsedexcitation line is controlled by TTL logic.
 23. The apparatus of claim21, wherein said pulsed excitation line is controlled by mechanical orelectronic means.
 24. The apparatus of claim 22, wherein said apparatusgenerates a sequence of discrete excitation lines that aretime-correlated with the fluorescence emission signals from the sample.25. The apparatus of claim 1, wherein at least one of the laserscomprises a diode laser.
 26. The apparatus of claim 1, wherein at leastone of the lasers comprises a semiconductor laser.
 27. The apparatus ofclaim 1, wherein at least one of the lasers comprises a gas laser. 28.The apparatus of claim 1, wherein at least one of the lasers comprises adiode pumped solid state laser.
 29. The apparatus of claim 28, whereinat least one of the solid state lasers comprises a Neodymium laser. 30.The apparatus of claim 1, further comprising a Raman shifter in operablerelation with at least one laser beam.
 31. The apparatus of claim 1,wherein the excitation wavelength provided by each laser is opticallymatched to the absorption wavelength of each fluorophore.
 32. Theapparatus of claim 1, wherein the detector comprises a charged coupledevice.
 33. The apparatus of claim 1, wherein the detector comprises aphotomultiplier tube.
 34. The apparatus of claim 1, wherein the detectorcomprises a silicon avalanche photodiode.
 35. The apparatus of claim 1,wherein the detector comprises a silicon PIN detector.
 36. The apparatusof claim 1, wherein the footprint of said device is less than 4 ft′×4ft′×2 ft.
 37. The apparatus of claim 36, wherein the footprint of saiddevice is less than 1 ft×1 ft×2 ft.
 38. The apparatus of claim 36,wherein the footprint of said device is less than 1-in×3-in×6-in.
 39. Amethod of identifying sample components comprising: (a) preparing asample comprising sample components, a first dye and a second dye; (b)placing the sample in the beam path of a first excitation line and asecond excitation line; (c) sequentially firing the first excitationline and the second excitation line; (d) collecting fluorescence signalsfrom the samples as a function of time; and (e) sorting the fluorescenceby each excitation line's on-time window, wherein the sample componentsare identified.
 40. The method of claim 39, wherein the fluorescencesignals are collected from discrete time periods in which no excitationline is incident on the sample, the time periods occurring between thefiring of the two excitation lines.
 41. The method of claim 39, whereinthe absorption maxima of the first dye substantially corresponds to theexcitation wavelength of the first excitation line.
 42. The method ofclaim 39, wherein the absorption maxima of the second dye substantiallycorresponds to the excitation wavelength of the second excitation line.43. The method of claim 42, further comprising a third and fourth dyeand a third and fourth excitation line, wherein the absorption maxima ofthe third and fourth dyes substantially correspond to the excitationwavelength of the third and four excitation lines.
 44. The method ofclaim 43, further comprising a fifth, sixth, seventh and eighth dye anda fifth, sixth, seventh and eighth excitation line, wherein theabsorption maxima of the fifth, sixth, seventh and eighth dyessubstantially correspond to the excitation wavelength of the fifth,sixth, seventh and eighth excitation lines.
 45. The method of claim 44,further comprising a ninth, tenth, eleventh, twelfth, thirteenth,fourteenth, fifteenth, and sixteenth excitation line, wherein theabsorption maxima of the ninth, tenth, eleventh, twelfth, thirteenth,fourteenth, fifteenth, and sixteenth dyes substantially correspond tothe excitation wavelength of the ninth, tenth, eleventh, twelfth,thirteenth, fourteenth, fifteenth, and sixteenth excitation lines. 46.The method of claim 39, wherein at least one of said dyes is a zanthene,fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene,phthalocyanine, phycobiliprotein, Alexa, or squariane dye.
 47. Themethod of claim 46, wherein at least one of said dyes is a BODIPY dye.48. The method of claim 39, wherein said sample components enable thedetermination of SNPs.
 49. The method of claim 48, wherein said methodis for the high-throughput identification of informative SNPs.
 50. Themethod of claim 48, wherein said SNPs are obtained directly from genomicDNA material.
 51. The method of claim 48, wherein said SNPs are obtainedfrom PCR amplified material.
 52. The method of claim 48, wherein saidSNPs are obtained from cloned material derived directly from genomic DNAmaterial or PCR amplified material.
 53. The method of claim 48, whereinsaid SNPs are obtained using a single nucleotide primer extensionmethod.
 54. The method of claim 50, wherein said single nucleotideprimer extension method comprises using single unlabeled dNTPs, singlelabeled dNTPs, single 3′-modified dNTPs, single base-modified 3′-dNTPs,single alpha-thio-dNTPs or single labeled 2′,3′-dideoxynucleotides 55.The method of claim 39, comprising a mini-sequencing method comprisesusing single unlabeled dNTPs, single labeled dNTPs, single 3′-modifieddNTPs, single base-modified 3′-dNTPs, single alpha-thio-dNTPs or singlelabeled 2′,3′-dideoxynucleotides.
 56. The method of claim 55, whereinsaid mini-sequencing method comprises an SNP.
 57. The method of claim56, wherein said mini-sequencing method comprises multiple SNPs.
 58. Themethod of claim 48, wherein said SNPs are obtained using Sangersequencing.
 59. The method of claim 48, wherein the analyzing of saidsignals is adapted for the accurate diagnosis of inherited disease,better prognosis of risk susceptibilities, identification of sporadicmutations, or prescribing tailor-made daily drug regimens for individualpatients.
 60. The method of claim 39, wherein the analyzing of saidsignals is adapted for routine usage in clinical diagnostics, forensicsapplications or determining general sequencing methodologies.
 61. Amethod of identifying sample components comprising: (a) obtaining abiological sample; (b) labeling said sample with one or morefluorophores; (c) separating components of said sample; and (d)detecting said sample components with a device wherein said devicecomprises: one or more lasers configured to emit two or more excitationlines, each excitation line having a different excitation wavelength; atiming circuit coupled to the one or more lasers and configured to firethe two or more excitation lines sequentially according to a timingprogram to produce time-correlated fluorescence emission signals fromthe sample; and a non-dispersive detector positioned to collect thetime-correlated fluorescence emission signals; wherein said detectorcollects time correlated data from said sample comprising fluorescentemissions of the sample as a result of irradiation by the one or moreexcitation lines.
 62. The method of claim 61, wherein said samplecomponents are nucleic acids.
 63. The method of claim 61, wherein saidsample components are amino acids.
 64. The method of claim 61, whereinsaid sample components are proteins.
 65. The method of claim 61, whereinsaid separating is by electrophoresis.
 66. The method of claim 61,wherein said separating is by chromatography.
 67. The method of claim61, wherein said separating is by mass spectrometry.
 68. The method ofclaim 61, wherein said sample components are addressed on high densitychip arrays.
 69. The method of claim 61, further comprising: (e)contacting said sample components on a surface comprising immobilizedoligonucleotides at known locations on said surface; and (f) performinga single nucleotide incorporation assay.
 70. The method of claim 61,further comprising: (e) contacting said sample components on a surfacecomprising immobilized oligonucleotides at known locations on saidsurface; and (f) performing a mini-sequencing assay.
 71. The method ofclaim 69, further comprising rastering said surface or said excitationlines such that said excitation lines contact said surface at multiplelocations.
 72. A device comprising: (a) one or more lasers having two ormore excitation lines; (b) one or more beam steering mirrors whereinsaid excitation lines each strike said mirrors; (c) a first prism,wherein said two or more excitation lines strike one surface and exitfrom a second surface of said first prism; and (d) a second prism at anangle relative to said first prism, wherein said two or more excitationlines strike one surface of said second prism after exiting said firstprism and exit said second prism, wherein said two or more excitationlines are substantially colinear or coaxial after exiting said secondprism.
 73. The method of claim 72, wherein said two or more excitationlines are substantially coaxial after exiting said second prism.
 74. Themethod of claim 72, wherein said two or more excitation lines aresubstantially colinear after exiting said second prism.
 75. A method ofilluminating a sample comprising: (a) steering two or more excitationlines onto a first surface of a first prism; (b) steering two or moreexcitation lines from the second surface of said first prism to a firstsurface of a second prism; wherein said second prism is angled about 45°from said first prism; (c) steering said two or more excitation linesonto a sample after exiting second surface of said second prism, whereinsaid two or more excitation lines are substantially colinear or coaxialafter exiting said second prism.
 76. The method of claim 75, whereinsaid two or more excitation lines are substantially coaxial afterexiting said second prism.
 77. The method of claim 75, wherein said twoor more excitation lines are substantially colinear after exiting saidsecond prism.
 78. A method of controlling a sequence of excitation linescomprising: obtaining a TTL circuit comprising an electronic stepperwherein said circuit is operationally connected to one or more lasershaving two or more excitation lines; and controlling the sequentialfiring of the one or more lasers having two or more excitation lineswith a clock pulse from the circuit, wherein the frequency of firing onelaser is equivalent to the frequency of firing a second laser, butphased shifted so that one or more lasers having two or more excitationlines can be sequentially pulsed.
 79. The method of claim 78, whereinthe cycle time of one clock pulse is from 1μsecond to 5 seconds.
 80. Themethod of claim 78, wherein the length of time a first laser produces anexcitation line is similar to the length of time a second laser producesan excitation line.
 81. The method of claim 78, wherein between 2-to-16excitation lines are sequentially pulsed.
 82. The method of claim 81,wherein between 2-to-8 excitation lines are sequentially pulsed.
 83. Amethod of controlling a sequence of excitation lines comprising:obtaining a TTL circuit comprising an electronic stepper wherein saidcircuit is operationally connected to two or more lasers; andcontrolling the sequential firing of the two or more lasers with a clockpulse from the circuit, wherein the frequency of firing a first laser isdifferent from the frequency of firing a second laser.
 84. The method ofclaim 83, comprising between 2-to-16 lasers.